##############################################################################
##############################################################################
###
### Running command:
###
###   /home/biocbuild/bbs-3.22-bioc/R/bin/R CMD check --install=check:FLAMES.install-out.txt --library=/home/biocbuild/bbs-3.22-bioc/R/site-library --timings FLAMES_2.3.5.tar.gz
###
##############################################################################
##############################################################################


* using log directory ‘/home/biocbuild/bbs-3.22-bioc/meat/FLAMES.Rcheck’
* using R version 4.5.1 Patched (2025-08-23 r88802)
* using platform: x86_64-pc-linux-gnu
* R was compiled by
    gcc (Ubuntu 13.3.0-6ubuntu2~24.04) 13.3.0
    GNU Fortran (Ubuntu 13.3.0-6ubuntu2~24.04) 13.3.0
* running under: Ubuntu 24.04.3 LTS
* using session charset: UTF-8
* checking for file ‘FLAMES/DESCRIPTION’ ... OK
* this is package ‘FLAMES’ version ‘2.3.5’
* package encoding: UTF-8
* checking package namespace information ... OK
* checking package dependencies ... INFO
Imports includes 47 non-default packages.
Importing from so many packages makes the package vulnerable to any of
them becoming unavailable.  Move as many as possible to Suggests and
use conditionally.
* checking if this is a source package ... OK
* checking if there is a namespace ... OK
* checking for hidden files and directories ... NOTE
Found the following hidden files and directories:
  .dockerignore
These were most likely included in error. See section ‘Package
structure’ in the ‘Writing R Extensions’ manual.
* checking for portable file names ... OK
* checking for sufficient/correct file permissions ... OK
* checking whether package ‘FLAMES’ can be installed ... NOTE
Found the following notes/warnings:
  Non-staged installation was used
See ‘/home/biocbuild/bbs-3.22-bioc/meat/FLAMES.Rcheck/00install.out’ for details.
* used C++ compiler: ‘g++ (Ubuntu 13.3.0-6ubuntu2~24.04) 13.3.0’
* checking C++ specification ... OK
* checking installed package size ... INFO
  installed size is  6.0Mb
  sub-directories of 1Mb or more:
    bin    1.1Mb
    data   1.8Mb
    libs   1.5Mb
* checking package directory ... OK
* checking ‘build’ directory ... OK
* checking DESCRIPTION meta-information ... OK
* checking top-level files ... OK
* checking for left-over files ... OK
* checking index information ... OK
* checking package subdirectories ... OK
* checking code files for non-ASCII characters ... OK
* checking R files for syntax errors ... OK
* checking whether the package can be loaded ... OK
* checking whether the package can be loaded with stated dependencies ... OK
* checking whether the package can be unloaded cleanly ... OK
* checking whether the namespace can be loaded with stated dependencies ... OK
* checking whether the namespace can be unloaded cleanly ... OK
* checking loading without being on the library search path ... OK
* checking dependencies in R code ... NOTE
There are ::: calls to the package's namespace in its code. A package
  almost never needs to use ::: for its own objects:
  'blaze' 'find_barcode' 'gene_quantification' 'isoform_identification'
  'minimap2_align' 'transcript_quantification'
* checking S3 generic/method consistency ... OK
* checking replacement functions ... OK
* checking foreign function calls ... OK
* checking R code for possible problems ... NOTE
BulkPipeline: no visible global function definition for ‘new’
BulkPipeline: no visible global function definition for ‘setNames’
MultiSampleSCPipeline: no visible global function definition for ‘new’
MultiSampleSCPipeline: no visible global function definition for
  ‘setNames’
SingleCellPipeline: no visible global function definition for ‘new’
SingleCellPipeline: no visible global function definition for
  ‘setNames’
addRowRanges: no visible global function definition for ‘head’
addRowRanges: no visible global function definition for ‘as’
add_gene_counts: no visible global function definition for ‘as’
cache_dir: no visible global function definition for ‘packageVersion’
chisq_test_by_gene: no visible global function definition for
  ‘chisq.test’
create_sce_from_dir: no visible global function definition for
  ‘setNames’
create_spe: no visible binding for global variable ‘barcode’
create_spe: no visible binding for global variable ‘in_tissue’
download_oarfish: no visible global function definition for
  ‘download.file’
download_oarfish: no visible global function definition for ‘unzip’
filter_coverage: no visible global function definition for
  ‘starts_with’
filter_coverage: no visible binding for global variable ‘filter_res’
find_variants: no visible global function definition for ‘setNames’
find_variants_grange: no visible binding for global variable
  ‘which_label’
find_variants_grange: no visible binding for global variable
  ‘nucleotide’
find_variants_grange: no visible binding for global variable ‘pos’
find_variants_grange: no visible binding for global variable ‘count’
find_variants_grange: no visible binding for global variable
  ‘counts_no_ins’
find_variants_grange: no visible binding for global variable ‘ref’
generate_sc_sce: no visible binding for global variable ‘FSM_match’
get_coverage: no visible binding for global variable ‘Freq’
homopolymer_pct : <anonymous>: no visible binding for global variable
  ‘Freq’
homopolymer_pct : <anonymous>: no visible binding for global variable
  ‘pct’
plot_coverage: no visible binding for global variable ‘tr_length’
plot_coverage: no visible binding for global variable ‘read_counts’
plot_coverage: no visible binding for global variable ‘total_counts’
plot_coverage: no visible binding for global variable ‘cumpct’
plot_coverage: no visible binding for global variable ‘length_bin’
plot_coverage: no visible binding for global variable ‘min_length’
plot_coverage: no visible binding for global variable ‘max_length’
plot_coverage: no visible global function definition for ‘head’
plot_coverage: no visible binding for global variable ‘transcript’
plot_demultiplex_raw: no visible binding for global variable ‘Sample’
plot_demultiplex_raw: no visible binding for global variable
  ‘CellBarcode’
plot_demultiplex_raw: no visible binding for global variable ‘UMI’
plot_demultiplex_raw: no visible binding for global variable
  ‘UMI_count’
plot_demultiplex_raw: no visible binding for global variable
  ‘barcode_rank’
plot_demultiplex_raw: no visible binding for global variable
  ‘FlankEditDist’
plot_demultiplex_raw: no visible binding for global variable ‘n_reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘BarcodeEditDist’
plot_demultiplex_raw: no visible binding for global variable ‘total
  reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘demultiplexed reads’
plot_demultiplex_raw: no visible binding for global variable ‘single
  match reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘undemultiplexted reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘multi-matching reads’
plot_demultiplex_raw: no visible binding for global variable ‘Type’
plot_demultiplex_raw: no visible binding for global variable ‘Reads’
plot_demultiplex_raw: no visible binding for global variable ‘input’
plot_demultiplex_raw: no visible binding for global variable ‘output’
plot_demultiplex_raw: no visible binding for global variable
  ‘read1_with_adapter’
plot_demultiplex_raw: no visible binding for global variable ‘Count’
plot_flagstat: no visible global function definition for ‘everything’
plot_flagstat: no visible binding for global variable ‘name’
plot_flagstat: no visible binding for global variable ‘value’
plot_isoform_reduced_dim: no visible binding for global variable ‘x’
plot_isoform_reduced_dim: no visible binding for global variable ‘y’
plot_isoform_reduced_dim: no visible binding for global variable ‘expr’
plot_spatial: no visible binding for global variable ‘imageX’
plot_spatial: no visible binding for global variable ‘imageY’
plot_spatial_feature: no visible binding for global variable ‘imageX’
plot_spatial_feature: no visible binding for global variable ‘imageY’
plot_spatial_feature: no visible binding for global variable ‘x’
plot_spatial_feature: no visible binding for global variable ‘y’
plot_spatial_feature: no visible global function definition for
  ‘scale_alpha_continuous’
plot_spatial_feature: no visible global function definition for
  ‘scale_colour_gradient’
plot_spatial_isoform: no visible global function definition for ‘head’
plot_spatial_pie: no visible global function definition for ‘setNames’
plot_spatial_pie: no visible global function definition for ‘head’
plot_spatial_pie: no visible binding for global variable ‘imageX’
plot_spatial_pie: no visible binding for global variable ‘imageY’
sc_gene_entropy: no visible global function definition for ‘as’
sc_genotype: no visible binding for global variable ‘allele’
sc_genotype: no visible binding for global variable ‘allele_count’
sc_genotype: no visible binding for global variable ‘barcode’
sc_genotype: no visible binding for global variable ‘pct’
sc_mutations: no visible binding for global variable ‘mutation_index’
sc_mutations: no visible binding for global variable ‘bam_index’
sc_plot_genotype: no visible global function definition for ‘setNames’
sc_plot_genotype: no visible binding for global variable ‘barcode’
sc_plot_genotype: no visible binding for global variable ‘genotype’
sc_plot_genotype: no visible binding for global variable ‘x’
sc_plot_genotype: no visible binding for global variable ‘y’
sc_transcript_usage_chisq: no visible global function definition for
  ‘as’
sc_transcript_usage_chisq: no visible binding for global variable
  ‘p.value’
sc_transcript_usage_chisq: no visible binding for global variable
  ‘adj.p.value’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘total’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘test’
sc_transcript_usage_permutation: no visible global function definition
  for ‘as’
sc_transcript_usage_permutation : <anonymous>: no visible global
  function definition for ‘as’
sc_transcript_usage_permutation : <anonymous> : <anonymous>: no visible
  global function definition for ‘na.omit’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘transcript’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘p.value’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘adj.p.value’
variant_count_tb: no visible binding for global variable ‘barcode’
variant_count_tb: no visible binding for global variable ‘allele_count’
variant_count_tb: no visible binding for global variable
  ‘cell_total_reads’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘expect_cell_number’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘fastq’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘demultiplexed_fastq’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘barcodes_file’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘outdir’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible global
  function definition for ‘setNames’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline : <anonymous>: no
  visible global function definition for ‘setNames’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘expect_cell_number’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘fastq’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘demultiplexed_fastq’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘barcodes_file’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘outdir’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible global
  function definition for ‘setNames’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘j’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘genome_bam’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘minimap2’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘samtools’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘threads’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘outdir’
plot_durations,FLAMES.Pipeline: no visible binding for global variable
  ‘step’
plot_durations,FLAMES.Pipeline: no visible binding for global variable
  ‘duration’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘j’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘transcriptome_assembly’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘transcriptome_bam’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘minimap2’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘samtools’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘outdir’
resume_FLAMES,FLAMES.Pipeline : <anonymous>: no visible global function
  definition for ‘capture.output’
run_FLAMES,FLAMES.Pipeline : <anonymous>: no visible global function
  definition for ‘capture.output’
Undefined global functions or variables:
  BarcodeEditDist CellBarcode Count FSM_match FlankEditDist Freq Reads
  Sample Type UMI UMI_count adj.p.value allele allele_count as
  bam_index barcode barcode_rank barcodes_file capture.output
  cell_total_reads chisq.test count counts_no_ins cumpct demultiplexed
  reads demultiplexed_fastq download.file duration everything
  expect_cell_number expr fastq filter_res genome_bam genotype head
  imageX imageY in_tissue input j length_bin max_length min_length
  minimap2 multi-matching reads mutation_index n_reads na.omit name new
  nucleotide outdir output p.value packageVersion pct pos
  read1_with_adapter read_counts ref samtools scale_alpha_continuous
  scale_colour_gradient setNames single match reads starts_with step
  test threads total total reads total_counts tr_length transcript
  transcriptome_assembly transcriptome_bam undemultiplexted reads unzip
  value which_label x y
Consider adding
  importFrom("base", "match", "single")
  importFrom("methods", "as", "new")
  importFrom("stats", "chisq.test", "na.omit", "setNames", "step")
  importFrom("utils", "capture.output", "download.file", "head",
             "packageVersion", "unzip")
to your NAMESPACE file (and ensure that your DESCRIPTION Imports field
contains 'methods').
* checking Rd files ... OK
* checking Rd metadata ... OK
* checking Rd cross-references ... OK
* checking for missing documentation entries ... OK
* checking for code/documentation mismatches ... OK
* checking Rd \usage sections ... OK
* checking Rd contents ... OK
* checking for unstated dependencies in examples ... OK
* checking contents of ‘data’ directory ... OK
* checking data for non-ASCII characters ... OK
* checking data for ASCII and uncompressed saves ... OK
* checking line endings in shell scripts ... OK
* checking line endings in C/C++/Fortran sources/headers ... OK
* checking line endings in Makefiles ... OK
* checking compilation flags in Makevars ... OK
* checking for GNU extensions in Makefiles ... INFO
GNU make is a SystemRequirements.
* checking for portable use of $(BLAS_LIBS) and $(LAPACK_LIBS) ... OK
* checking use of PKG_*FLAGS in Makefiles ... OK
* checking include directives in Makefiles ... OK
* checking compiled code ... NOTE
Note: information on .o files is not available
File ‘/home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/libs/FLAMES.so’:
  Found ‘abort’, possibly from ‘abort’ (C)
  Found ‘exit’, possibly from ‘exit’ (C)
  Found ‘stderr’, possibly from ‘stderr’ (C)
  Found ‘stdout’, possibly from ‘stdout’ (C)

Compiled code should not call entry points which might terminate R nor
write to stdout/stderr instead of to the console, nor use Fortran I/O
nor system RNGs nor [v]sprintf. The detected symbols are linked into
the code but might come from libraries and not actually be called.

See ‘Writing portable packages’ in the ‘Writing R Extensions’ manual.
* checking files in ‘vignettes’ ... OK
* checking examples ... OK
Examples with CPU (user + system) or elapsed time > 5s
                               user system elapsed
plot_isoform_reduced_dim     23.020  0.127  23.146
blaze                         4.266 17.202  12.146
find_variants                18.922  0.072  18.389
bulk_long_pipeline            2.282 12.457   2.416
sc_long_multisample_pipeline  7.916  6.197   7.962
sc_plot_genotype             10.591  0.816  10.266
MultiSampleSCPipeline         9.612  0.609  10.629
sc_DTU_analysis               6.960  1.844   6.877
plot_isoform_heatmap          6.839  0.092   6.931
create_sce_from_dir           3.445  2.529   3.658
sc_long_pipeline              3.145  1.962   2.884
* checking for unstated dependencies in ‘tests’ ... OK
* checking tests ...
  Running ‘testthat.R’
 OK
* checking for unstated dependencies in vignettes ... OK
* checking package vignettes ... OK
* checking re-building of vignette outputs ... OK
* checking PDF version of manual ... OK
* DONE

Status: 5 NOTEs
See
  ‘/home/biocbuild/bbs-3.22-bioc/meat/FLAMES.Rcheck/00check.log’
for details.

##############################################################################
##############################################################################
###
### Running command:
###
###   /home/biocbuild/bbs-3.22-bioc/R/bin/R CMD INSTALL FLAMES
###
##############################################################################
##############################################################################


* installing to library ‘/home/biocbuild/bbs-3.22-bioc/R/site-library’
* installing *source* package ‘FLAMES’ ...
** this is package ‘FLAMES’ version ‘2.3.5’
** using non-staged installation via StagedInstall field
** libs
using C++ compiler: ‘g++ (Ubuntu 13.3.0-6ubuntu2~24.04) 13.3.0’
using C++17
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c RcppExports.cpp -o RcppExports.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c RcppFunctions.cpp -o RcppFunctions.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/BamRecord.cpp -o classes/BamRecord.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/GFFRecord.cpp -o classes/GFFRecord.o
In file included from classes/GFFRecord.cpp:8:
classes/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
classes/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/GeneAnnotationParser.cpp -o classes/GeneAnnotationParser.o
In file included from classes/GeneAnnotationParser.cpp:15:
classes/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
classes/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/Isoforms.cpp -o classes/Isoforms.o
In file included from classes/Isoforms.cpp:16:
classes/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
classes/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
classes/Isoforms.cpp: In member function ‘void Isoforms::update_all_splice()’:
classes/Isoforms.cpp:233:35: warning: comparison of integer expressions of different signedness: ‘std::vector<StartEndPair>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  233 |                 if (blocks.size() >= (int)this->Min_sup_cnt) {
      |                     ~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp: In member function ‘void Isoforms::filter_TSS_TES(std::ofstream&, DoubleJunctions, float)’:
classes/Isoforms.cpp:368:33: warning: comparison of integer expressions of different signedness: ‘std::unordered_map<int, int>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  368 |         if ((left_counts.size() < (int)this->Min_sup_cnt) ||
      |              ~~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp:369:38: warning: comparison of integer expressions of different signedness: ‘std::unordered_map<int, int>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  369 |                 (right_counts.size() < (int)this->Min_sup_cnt)) {
      |                  ~~~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp: In member function ‘void Isoforms::match_known_annotation(const std::unordered_map<std::__cxx11::basic_string<char>, Junctions>&, const std::unordered_map<std::__cxx11::basic_string<char>, Pos>&, const std::unordered_map<std::__cxx11::basic_string<char>, std::vector<StartEndPair> >&, GeneBlocks, std::unordered_map<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> >)’:
classes/Isoforms.cpp:662:51: warning: comparison of integer expressions of different signedness: ‘int’ and ‘const long unsigned int’ [-Wsign-compare]
  662 |                                 for (int j = 0; j < std::min(raw_iso_key.size() - 1, junction.junctions.size()); j++) {
      |                                                 ~~^~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp:713:43: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<int>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  713 |                         for (int i = 0; i < new_exons.size(); ++i) {
      |                                         ~~^~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp:725:46: warning: comparisons like ‘X<=Y<=Z’ do not have their mathematical meaning [-Wparentheses]
  725 |                                 } else if (0 < i < raw_iso_key.size() - 1) { // a site from the middle somewhere
      |                                            ~~^~~
classes/Isoforms.cpp: In member function ‘std::string Isoforms::isoform_to_gff3(float)’:
classes/Isoforms.cpp:1140:43: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<int>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
 1140 |                         for (int i = 0; i < exons.size(); i+=2) {
      |                                         ~~^~~~~~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/junctions.cpp -o classes/junctions.o
In file included from classes/junctions.cpp:12:
classes/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
classes/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
classes/junctions.cpp: In function ‘std::unordered_map<std::__cxx11::basic_string<char>, std::vector<StartEndPair> > get_gene_flat(const std::unordered_map<std::__cxx11::basic_string<char>, std::vector<std::__cxx11::basic_string<char> > >&, const std::unordered_map<std::__cxx11::basic_string<char>, std::vector<StartEndPair> >&)’:
classes/junctions.cpp:166:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<StartEndPair>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  166 |         for (int i = 1; i < exons.size(); i++) {
      |                         ~~^~~~~~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/find_isoform.cpp -o main-functions/find_isoform.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/flexiplex.cpp -o main-functions/flexiplex.o
main-functions/flexiplex.cpp: In function ‘unsigned int edit_distance(const std::string&, const std::string&, unsigned int&, int)’:
main-functions/flexiplex.cpp:123:21: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘int’ [-Wsign-compare]
  123 |       if (min_value <= max_editd)
      |           ~~~~~~~~~~^~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘std::string get_umi(const std::string&, const std::vector<std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > >&, const std::vector<int>&, int, int, bool, int, int)’:
main-functions/flexiplex.cpp:164:22: warning: comparison of integer expressions of different signedness: ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  164 |     if (seq.length() < umi_start + umi_length) {
      |         ~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:186:36: warning: comparison of integer expressions of different signedness: ‘std::vector<int>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  186 |     if (read_to_subpatterns.size() > umi_index + 1) {
      |         ~~~~~~~~~~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘Barcode get_barcode(const std::string&, const std::unordered_set<std::__cxx11::basic_string<char> >&, int, int, const std::vector<std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > >&)’:
main-functions/flexiplex.cpp:299:19: warning: comparison of integer expressions of different signedness: ‘int’ and ‘__gnu_cxx::__alloc_traits<std::allocator<long unsigned int>, long unsigned int>::value_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  299 |     if (i_pattern >= subpattern_ends[i_subpattern]) {
main-functions/flexiplex.cpp:358:22: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘int’ [-Wsign-compare]
  358 |     if (editDistance == barcode.editd) {
      |         ~~~~~~~~~~~~~^~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:360:29: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘int’ [-Wsign-compare]
  360 |     } else if (editDistance < barcode.editd &&
      |                ~~~~~~~~~~~~~^~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:361:29: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘int’ [-Wsign-compare]
  361 |                editDistance <= barcode_max_editd) { // if best so far, update
      |                ~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘std::vector<Barcode> big_barcode_search(const std::string&, const std::unordered_set<std::__cxx11::basic_string<char> >&, int, int, const std::vector<std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > >&)’:
main-functions/flexiplex.cpp:400:29: warning: comparison of integer expressions of different signedness: ‘const int’ and ‘const std::__cxx11::basic_string<char>::size_type’ {aka ‘const long unsigned int’} [-Wsign-compare]
  400 |       if (barcode.flank_end == std::string::npos) {
      |           ~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘void write_bgzfstring(BGZF*, const std::string&)’:
main-functions/flexiplex.cpp:423:21: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  423 |   if (bytes_written != str.size()) {
      |       ~~~~~~~~~~~~~~^~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘void print_stats(const std::string&, const std::vector<Barcode>&, BGZF*)’:
main-functions/flexiplex.cpp:438:28: warning: comparison of integer expressions of different signedness: ‘const int’ and ‘const std::__cxx11::basic_string<char>::size_type’ {aka ‘const long unsigned int’} [-Wsign-compare]
  438 |         (barcode.flank_end == std::string::npos ? "True" : "False") + "\n";
      |          ~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘void print_read(const std::string&, const std::string&, const std::string&, const std::vector<Barcode>&, BGZF*, std::unordered_set<std::__cxx11::basic_string<char> >&, bool, bool, bool)’:
main-functions/flexiplex.cpp:475:21: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<Barcode>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  475 |   for (int b = 0; b < vec_bc.size(); b++) {
      |                   ~~^~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:490:32: warning: comparison of integer expressions of different signedness: ‘const int’ and ‘const std::__cxx11::basic_string<char>::size_type’ {aka ‘const long unsigned int’} [-Wsign-compare]
  490 |     if (vec_bc.at(b).flank_end == std::string::npos) {
      |         ~~~~~~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:495:23: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<Barcode>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  495 |     for (int f = 0; f < vec_bc.size(); f++) {
      |                     ~~^~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘Rcpp::IntegerVector flexiplex_cpp(Rcpp::StringVector, Rcpp::String, bool, int, int, Rcpp::StringVector, Rcpp::String, Rcpp::String, Rcpp::String, bool, int)’:
main-functions/flexiplex.cpp:754:25: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<std::vector<SearchResult> >::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  754 |       for (int t = 0; t < sr_v.size();
      |                       ~~^~~~~~~~~~~~~
main-functions/flexiplex.cpp:759:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<SearchResult>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  759 |         for (int r = 0; r < sr_v[t].size(); r++) { // loop over the reads
      |                         ~~^~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:761:29: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<Barcode>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  761 |           for (int b = 0; b < sr_v[t][r].vec_bc_for.size(); b++)
      |                           ~~^~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:763:29: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<Barcode>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  763 |           for (int b = 0; b < sr_v[t][r].vec_bc_rev.size(); b++)
      |                           ~~^~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/get_transcript_seq.cpp -o main-functions/get_transcript_seq.o
main-functions/get_transcript_seq.cpp: In function ‘void write_fa(const std::string&, const std::string&, const std::string&, int)’:
main-functions/get_transcript_seq.cpp:87:14: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
   87 |     while (i < seq.size()) {
      |            ~~^~~~~~~~~~~~
main-functions/get_transcript_seq.cpp:88:26: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
   88 |         if (i + wrap_len > seq.size()) {
      |             ~~~~~~~~~~~~~^~~~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/group_bam2isoform.cpp -o main-functions/group_bam2isoform.o
In file included from main-functions/group_bam2isoform.cpp:18:
main-functions/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
main-functions/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/pileup_readid.cpp -o main-functions/pileup_readid.o
main-functions/pileup_readid.cpp: In function ‘Rcpp::NumericMatrix variant_count_matrix_cpp(Rcpp::String, Rcpp::String, int, bool, bool)’:
main-functions/pileup_readid.cpp:268:21: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  268 |   for (int i = 0; i < BASES.size(); i++) {
      |                   ~~^~~~~~~~~~~~~~
main-functions/pileup_readid.cpp: In instantiation of ‘std::unordered_map<std::__cxx11::basic_string<char>, std::vector<std::vector<std::__cxx11::basic_string<char> > > > group_umis(const TableType&, int) [with TableType = std::unordered_map<std::__cxx11::basic_string<char>, std::unordered_map<std::__cxx11::basic_string<char>, std::unordered_map<std::__cxx11::basic_string<char>, unsigned int> > >]’:
main-functions/pileup_readid.cpp:342:30:   required from here
main-functions/pileup_readid.cpp:86:16: warning: unused variable ‘end’ [-Wunused-variable]
   86 |   unsigned int end;
      |                ^~~
main-functions/pileup_readid.cpp: In instantiation of ‘std::unordered_map<std::__cxx11::basic_string<char>, std::vector<std::vector<std::__cxx11::basic_string<char> > > > group_umis(const TableType&, int) [with TableType = std::unordered_map<std::__cxx11::basic_string<char>, std::unordered_map<std::__cxx11::basic_string<char>, std::array<unsigned int, 5> > >]’:
main-functions/pileup_readid.cpp:344:30:   required from here
main-functions/pileup_readid.cpp:86:16: warning: unused variable ‘end’ [-Wunused-variable]
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c tests/test-junctions.cpp -o tests/test-junctions.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c tests/test-parsing.cpp -o tests/test-parsing.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c utility/cigars.cpp -o utility/cigars.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c utility/edlib-1.2.7/edlib.cpp -o utility/edlib-1.2.7/edlib.o
gcc -std=gnu2x -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security -c utility/bam.c -o utility/bam.o
g++ -std=gnu++17 -shared -L/home/biocbuild/bbs-3.22-bioc/R/lib -L/usr/local/lib -o FLAMES.so RcppExports.o RcppFunctions.o classes/BamRecord.o classes/GFFRecord.o classes/GeneAnnotationParser.o classes/Isoforms.o classes/junctions.o main-functions/find_isoform.o main-functions/flexiplex.o main-functions/get_transcript_seq.o main-functions/group_bam2isoform.o main-functions/pileup_readid.o tests/test-junctions.o tests/test-parsing.o utility/cigars.o utility/edlib-1.2.7/edlib.o utility/bam.o -pthread -lz /home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/usrlib/libhts.a -lcurl -lbz2 -llzma -lz -L/home/biocbuild/bbs-3.22-bioc/R/lib -lR
if test -e "/usr/bin/strip" & test -e "/bin/uname" & [[ `uname` == "Linux" ]] ; then /usr/bin/strip --strip-debug FLAMES.so; fi
Building for x86_64
(cd submodule/minimap2 && make -f Makefile CFLAGS="-g -O2  -Wall -Werror=format-security -Wno-unused-result" minimap2)
make[1]: Entering directory '/home/biocbuild/bbs-3.22-bioc/meat/FLAMES/src/submodule/minimap2'
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  main.c -o main.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  kthread.c -o kthread.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  kalloc.c -o kalloc.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  misc.c -o misc.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  bseq.c -o bseq.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  sketch.c -o sketch.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  sdust.c -o sdust.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  options.c -o options.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  index.c -o index.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  lchain.c -o lchain.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  align.c -o align.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  hit.c -o hit.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  seed.c -o seed.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  jump.c -o jump.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  map.c -o map.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  format.c -o format.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  pe.c -o pe.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  esterr.c -o esterr.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  splitidx.c -o splitidx.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse2 -DHAVE_KALLOC  ksw2_ll_sse.c -o ksw2_ll_sse.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_extz2_sse.c -o ksw2_extz2_sse41.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_extd2_sse.c -o ksw2_extd2_sse41.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_exts2_sse.c -o ksw2_exts2_sse41.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse2 -mno-sse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH -DKSW_SSE2_ONLY  ksw2_extz2_sse.c -o ksw2_extz2_sse2.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse2 -mno-sse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH -DKSW_SSE2_ONLY  ksw2_extd2_sse.c -o ksw2_extd2_sse2.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse2 -mno-sse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH -DKSW_SSE2_ONLY  ksw2_exts2_sse.c -o ksw2_exts2_sse2.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_dispatch.c -o ksw2_dispatch.o
ar -csru libminimap2.a kthread.o kalloc.o misc.o bseq.o sketch.o sdust.o options.o index.o lchain.o align.o hit.o seed.o jump.o map.o format.o pe.o esterr.o splitidx.o ksw2_ll_sse.o ksw2_extz2_sse41.o ksw2_extd2_sse41.o ksw2_exts2_sse41.o ksw2_extz2_sse2.o ksw2_extd2_sse2.o ksw2_exts2_sse2.o ksw2_dispatch.o
ar: `u' modifier ignored since `D' is the default (see `U')
cc -g -O2  -Wall -Werror=format-security -Wno-unused-result main.o -o minimap2 -L. -lminimap2 -lm -lz -lpthread
make[1]: Leaving directory '/home/biocbuild/bbs-3.22-bioc/meat/FLAMES/src/submodule/minimap2'
echo "Installing binary to /home/biocbuild/bbs-3.22-bioc/meat/FLAMES/src/../inst/bin"
Installing binary to /home/biocbuild/bbs-3.22-bioc/meat/FLAMES/src/../inst/bin
mkdir -p ../inst/bin
cp submodule/minimap2/minimap2 ../inst/bin/
Rust version too old for edition 2024, skipping oarfish installation.
installing to /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/libs
** R
** data
** inst
** byte-compile and prepare package for lazy loading
** help
*** installing help indices
*** copying figures
** building package indices
** installing vignettes
** testing if installed package can be loaded
* DONE (FLAMES)

R version 4.5.1 Patched (2025-08-23 r88802) -- "Great Square Root"
Copyright (C) 2025 The R Foundation for Statistical Computing
Platform: x86_64-pc-linux-gnu

R is free software and comes with ABSOLUTELY NO WARRANTY.
You are welcome to redistribute it under certain conditions.
Type 'license()' or 'licence()' for distribution details.

R is a collaborative project with many contributors.
Type 'contributors()' for more information and
'citation()' on how to cite R or R packages in publications.

Type 'demo()' for some demos, 'help()' for on-line help, or
'help.start()' for an HTML browser interface to help.
Type 'q()' to quit R.

> # This file is part of the standard setup for testthat.
> # It is recommended that you do not modify it.
> #
> # Where should you do additional test configuration?
> # Learn more about the roles of various files in:
> # * https://r-pkgs.org/tests.html
> # * https://testthat.r-lib.org/reference/test_package.html#special-files
> 
> library(testthat)
> library(FLAMES)
> 
> test_check("FLAMES")
Writing configuration parameters to:  /tmp/Rtmppx9ZHJ/file1cf0f960c21471/config_file_1896697.json 
Writing configuration parameters to:  /tmp/Rtmppx9ZHJ/file1cf0f960c21471/config_file_1896697.json 
Writing configuration parameters to:  /tmp/Rtmppx9ZHJ/file1cf0f960c21471/config_file_1896697.json 
Writing configuration parameters to:  /tmp/Rtmppx9ZHJ/file1cf0f96dc9697b/config_file_1896697.json 
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmppx9ZHJ/file1cf0f9529b46e4/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
Skipping TSO trimming...
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmppx9ZHJ/file1cf0f96697418f/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
Skipping TSO trimming...
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmppx9ZHJ/file1cf0f96697418f/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmppx9ZHJ/file1cf0f92c8bbfe7/musc_rps24_1.fastq
Searching for barcodes...
Processing file: /tmp/Rtmppx9ZHJ/file1cf0f92c8bbfe7/musc_rps24_2.fastq
Searching for barcodes...
Processing file: /tmp/Rtmppx9ZHJ/file1cf0f92c8bbfe7/musc_rps24_3.fastq
Searching for barcodes...
Processing file: /tmp/Rtmppx9ZHJ/file1cf0f92c8bbfe7/musc_rps24_4.fastq
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
Skipping TSO trimming...
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmppx9ZHJ/file1cf0f94196a9e9/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
Skipping TSO trimming...
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmppx9ZHJ/file1cf0f91a8e7e72/config_file_1896697.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Mon Oct 20 00:03:15 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/Rtmppx9ZHJ/file1cf0f91a8e7e72/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/Rtmppx9ZHJ/file1cf0f91a8e7e72/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/Rtmppx9ZHJ/file1cf0f91a8e7e72/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Mon Oct 20 00:03:16 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
  |                                                                      |   0%[00:03:22] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

    https://xgboost.readthedocs.io/en/latest/tutorials/saving_model.html

  for more details about differences between saving model and serializing.

[00:03:22] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

    https://xgboost.readthedocs.io/en/latest/tutorials/saving_model.html

  for more details about differences between saving model and serializing.

[00:03:23] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

    https://xgboost.readthedocs.io/en/latest/tutorials/saving_model.html

  for more details about differences between saving model and serializing.

[00:03:23] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

    https://xgboost.readthedocs.io/en/latest/tutorials/saving_model.html

  for more details about differences between saving model and serializing.

[00:03:24] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

    https://xgboost.readthedocs.io/en/latest/tutorials/saving_model.html

  for more details about differences between saving model and serializing.

[00:03:24] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

    https://xgboost.readthedocs.io/en/latest/tutorials/saving_model.html

  for more details about differences between saving model and serializing.


  |                                                                            
  |=======================                                               |  33%
  |                                                                            
  |===============================================                       |  67%
  |                                                                            
  |======================================================================| 100%

Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Mon Oct 20 00:03:39 2025 -------------------
Realigning sample sample1 -> /tmp/Rtmppx9ZHJ/file1cf0f91a8e7e72/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/Rtmppx9ZHJ/file1cf0f91a8e7e72/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/Rtmppx9ZHJ/file1cf0f91a8e7e72/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Mon Oct 20 00:03:40 2025 ----------
2025-10-20T04:03:40.482111Z  INFO oarfish: setting user-provided filter parameters.
2025-10-20T04:03:40.482490Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmppx9ZHJ/file1cf0f91a8e7e72/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-10-20T04:03:40.482501Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-20T04:03:40.482504Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-20T04:03:40.482555Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-20T04:03:40.482561Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-20T04:03:40.484151Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-20T04:03:40.484266Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 253   │
│ aligned fraction too low        │ 4     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 1     │
│ reads with valid best alignment │ 96    │
╰─────────────────────────────────┴───────╯

2025-10-20T04:03:40.484288Z  INFO oarfish::bulk: Total number of alignment records : 116
2025-10-20T04:03:40.484301Z  INFO oarfish::bulk: number of aligned reads : 96
2025-10-20T04:03:40.484303Z  INFO oarfish::bulk: number of unique alignments : 86
2025-10-20T04:03:40.484881Z  INFO oarfish: oarfish completed successfully.
2025-10-20T04:03:40.491299Z  INFO oarfish: setting user-provided filter parameters.
2025-10-20T04:03:40.491678Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmppx9ZHJ/file1cf0f91a8e7e72/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-10-20T04:03:40.491685Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-20T04:03:40.491687Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-20T04:03:40.491739Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-20T04:03:40.491744Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-20T04:03:40.493263Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-20T04:03:40.493365Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 251   │
│ aligned fraction too low        │ 5     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 2     │
│ reads with valid best alignment │ 95    │
╰─────────────────────────────────┴───────╯

2025-10-20T04:03:40.493392Z  INFO oarfish::bulk: Total number of alignment records : 114
2025-10-20T04:03:40.493394Z  INFO oarfish::bulk: number of aligned reads : 95
2025-10-20T04:03:40.493396Z  INFO oarfish::bulk: number of unique alignments : 82
2025-10-20T04:03:40.493928Z  INFO oarfish: oarfish completed successfully.
2025-10-20T04:03:40.501272Z  INFO oarfish: setting user-provided filter parameters.
2025-10-20T04:03:40.501615Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmppx9ZHJ/file1cf0f91a8e7e72/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-10-20T04:03:40.501623Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-20T04:03:40.501626Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-20T04:03:40.501683Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-20T04:03:40.501688Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-20T04:03:40.504429Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2025-10-20T04:03:40.504673Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 398   │
│ aligned fraction too low        │ 12    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 5     │
│ reads with valid best alignment │ 179   │
╰─────────────────────────────────┴───────╯

2025-10-20T04:03:40.504709Z  INFO oarfish::bulk: Total number of alignment records : 239
2025-10-20T04:03:40.504712Z  INFO oarfish::bulk: number of aligned reads : 179
2025-10-20T04:03:40.504714Z  INFO oarfish::bulk: number of unique alignments : 143
2025-10-20T04:03:40.505346Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmppx9ZHJ/file1cf0f9dd4be/config_file_1896697.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Mon Oct 20 00:03:40 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/Rtmppx9ZHJ/file1cf0f9dd4be/sample1_align2genome.bam
sample2 ->/tmp/Rtmppx9ZHJ/file1cf0f9dd4be/sample2_align2genome.bam
sample3 ->/tmp/Rtmppx9ZHJ/file1cf0f9dd4be/sample3_align2genome.bam
-- Running step: isoform_identification @ Mon Oct 20 00:04:00 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
  |                                                                      |   0%
  |                                                                            
  |=======================                                               |  33%
  |                                                                            
  |===============================================                       |  67%
  |                                                                            
  |======================================================================| 100%

Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Mon Oct 20 00:04:19 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/Rtmppx9ZHJ/file1cf0f9dd4be/sample1_realign2transcript.bam
sample2 ->/tmp/Rtmppx9ZHJ/file1cf0f9dd4be/sample2_realign2transcript.bam
sample3 ->/tmp/Rtmppx9ZHJ/file1cf0f9dd4be/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Mon Oct 20 00:04:37 2025 ----------
2025-10-20T04:04:37.034383Z  INFO oarfish: setting user-provided filter parameters.
2025-10-20T04:04:37.034899Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmppx9ZHJ/file1cf0f9dd4be/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-10-20T04:04:37.034921Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-20T04:04:37.034925Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-20T04:04:37.034986Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-20T04:04:37.034991Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-20T04:04:37.036796Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-20T04:04:37.036919Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 253   │
│ aligned fraction too low        │ 4     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 1     │
│ reads with valid best alignment │ 96    │
╰─────────────────────────────────┴───────╯

2025-10-20T04:04:37.036939Z  INFO oarfish::bulk: Total number of alignment records : 116
2025-10-20T04:04:37.036942Z  INFO oarfish::bulk: number of aligned reads : 96
2025-10-20T04:04:37.036944Z  INFO oarfish::bulk: number of unique alignments : 86
2025-10-20T04:04:37.037520Z  INFO oarfish: oarfish completed successfully.
2025-10-20T04:04:37.048264Z  INFO oarfish: setting user-provided filter parameters.
2025-10-20T04:04:37.048724Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmppx9ZHJ/file1cf0f9dd4be/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-10-20T04:04:37.048733Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-20T04:04:37.048736Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-20T04:04:37.048792Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-20T04:04:37.048797Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-20T04:04:37.050415Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-20T04:04:37.050531Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 251   │
│ aligned fraction too low        │ 5     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 2     │
│ reads with valid best alignment │ 95    │
╰─────────────────────────────────┴───────╯

2025-10-20T04:04:37.050555Z  INFO oarfish::bulk: Total number of alignment records : 114
2025-10-20T04:04:37.050566Z  INFO oarfish::bulk: number of aligned reads : 95
2025-10-20T04:04:37.050568Z  INFO oarfish::bulk: number of unique alignments : 82
2025-10-20T04:04:37.051145Z  INFO oarfish: oarfish completed successfully.
2025-10-20T04:04:37.061838Z  INFO oarfish: setting user-provided filter parameters.
2025-10-20T04:04:37.062192Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmppx9ZHJ/file1cf0f9dd4be/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-10-20T04:04:37.062203Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-20T04:04:37.062206Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-20T04:04:37.062257Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-20T04:04:37.062263Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-20T04:04:37.064988Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2025-10-20T04:04:37.065150Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 398   │
│ aligned fraction too low        │ 12    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 5     │
│ reads with valid best alignment │ 179   │
╰─────────────────────────────────┴───────╯

2025-10-20T04:04:37.065178Z  INFO oarfish::bulk: Total number of alignment records : 239
2025-10-20T04:04:37.065181Z  INFO oarfish::bulk: number of aligned reads : 179
2025-10-20T04:04:37.065183Z  INFO oarfish::bulk: number of unique alignments : 143
2025-10-20T04:04:37.065850Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmppx9ZHJ/file1cf0f95c1afbbb/config_file_1896697.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Mon Oct 20 00:04:37 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/Rtmppx9ZHJ/file1cf0f95c1afbbb/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/Rtmppx9ZHJ/file1cf0f95c1afbbb/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/Rtmppx9ZHJ/file1cf0f95c1afbbb/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Mon Oct 20 00:04:38 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
  |                                                                      |   0%
  |                                                                            
  |=======================                                               |  33%
  |                                                                            
  |===============================================                       |  67%
  |                                                                            
  |======================================================================| 100%

Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Mon Oct 20 00:04:55 2025 -------------------
Realigning sample sample1 -> /tmp/Rtmppx9ZHJ/file1cf0f95c1afbbb/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/Rtmppx9ZHJ/file1cf0f95c1afbbb/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/Rtmppx9ZHJ/file1cf0f95c1afbbb/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Mon Oct 20 00:04:56 2025 ----------
00:04:56 Mon Oct 20 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmppx9ZHJ/file1cf0f94afa66ce/config_file_1896697.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Mon Oct 20 00:04:57 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/Rtmppx9ZHJ/file1cf0f94afa66ce/sample1_align2genome.bam
sample2 ->/tmp/Rtmppx9ZHJ/file1cf0f94afa66ce/sample2_align2genome.bam
sample3 ->/tmp/Rtmppx9ZHJ/file1cf0f94afa66ce/sample3_align2genome.bam
-- Running step: isoform_identification @ Mon Oct 20 00:05:15 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
  |                                                                      |   0%
  |                                                                            
  |=======================                                               |  33%
  |                                                                            
  |===============================================                       |  67%
  |                                                                            
  |======================================================================| 100%

Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Mon Oct 20 00:05:32 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/Rtmppx9ZHJ/file1cf0f94afa66ce/sample1_realign2transcript.bam
sample2 ->/tmp/Rtmppx9ZHJ/file1cf0f94afa66ce/sample2_realign2transcript.bam
sample3 ->/tmp/Rtmppx9ZHJ/file1cf0f94afa66ce/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Mon Oct 20 00:05:50 2025 ----------
00:05:50 Mon Oct 20 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Inputs:  ['/tmp/Rtmppx9ZHJ/file1cf0f95c1afbbb/sample1_realign2transcript.bam', '/tmp/Rtmppx9ZHJ/file1cf0f95c1afbbb/sample2_realign2transcript.bam', '/tmp/Rtmppx9ZHJ/file1cf0f95c1afbbb/sample3_realign2transcript.bam'] /tmp/Rtmppx9ZHJ/file1cf0f95c1afbbb/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 375, 'not_enough_coverage': 16, 'unmapped': 2})
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmppx9ZHJ/file1cf0f963ebb802/config_file_1896697.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Mon Oct 20 00:05:51 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/Rtmppx9ZHJ/file1cf0f963ebb802/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/Rtmppx9ZHJ/file1cf0f963ebb802/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/Rtmppx9ZHJ/file1cf0f963ebb802/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Mon Oct 20 00:05:52 2025 -------------
Inputs:  ['/tmp/Rtmppx9ZHJ/file1cf0f94afa66ce/sample1_realign2transcript.bam', '/tmp/Rtmppx9ZHJ/file1cf0f94afa66ce/sample2_realign2transcript.bam', '/tmp/Rtmppx9ZHJ/file1cf0f94afa66ce/sample3_realign2transcript.bam'] /tmp/Rtmppx9ZHJ/file1cf0f94afa66ce/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 375, 'not_enough_coverage': 16, 'unmapped': 2})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Oct 20 00:05:52 2025 -------------------
Realigning sample sample1 -> /tmp/Rtmppx9ZHJ/file1cf0f963ebb802/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/Rtmppx9ZHJ/file1cf0f963ebb802/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/Rtmppx9ZHJ/file1cf0f963ebb802/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Mon Oct 20 00:05:53 2025 ----------
2025-10-20T04:05:53.276135Z  INFO oarfish: setting user-provided filter parameters.
2025-10-20T04:05:53.276597Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmppx9ZHJ/file1cf0f963ebb802/sample1_realign2transcript.bam, contains 10 reference sequences.
2025-10-20T04:05:53.276607Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-20T04:05:53.276612Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-20T04:05:53.276679Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-20T04:05:53.276687Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-20T04:05:53.279412Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-20T04:05:53.279543Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 707   │
│ aligned fraction too low        │ 2     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 98    │
╰─────────────────────────────────┴───────╯

2025-10-20T04:05:53.279563Z  INFO oarfish::bulk: Total number of alignment records : 125
2025-10-20T04:05:53.279565Z  INFO oarfish::bulk: number of aligned reads : 98
2025-10-20T04:05:53.279568Z  INFO oarfish::bulk: number of unique alignments : 86
2025-10-20T04:05:53.280172Z  INFO oarfish: oarfish completed successfully.
2025-10-20T04:05:53.287283Z  INFO oarfish: setting user-provided filter parameters.
2025-10-20T04:05:53.287630Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmppx9ZHJ/file1cf0f963ebb802/sample2_realign2transcript.bam, contains 10 reference sequences.
2025-10-20T04:05:53.287638Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-20T04:05:53.287641Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-20T04:05:53.287708Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-20T04:05:53.287715Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-20T04:05:53.290419Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-20T04:05:53.290545Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 701   │
│ aligned fraction too low        │ 3     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 97    │
╰─────────────────────────────────┴───────╯

2025-10-20T04:05:53.290568Z  INFO oarfish::bulk: Total number of alignment records : 136
2025-10-20T04:05:53.290571Z  INFO oarfish::bulk: number of aligned reads : 97
2025-10-20T04:05:53.290573Z  INFO oarfish::bulk: number of unique alignments : 79
2025-10-20T04:05:53.291165Z  INFO oarfish: oarfish completed successfully.
2025-10-20T04:05:53.298042Z  INFO oarfish: setting user-provided filter parameters.
2025-10-20T04:05:53.298391Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmppx9ZHJ/file1cf0f963ebb802/sample3_realign2transcript.bam, contains 10 reference sequences.
2025-10-20T04:05:53.298401Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-20T04:05:53.298404Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-20T04:05:53.298466Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-20T04:05:53.298472Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-20T04:05:53.302763Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-20T04:05:53.302931Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 1060  │
│ aligned fraction too low        │ 6     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 187   │
╰─────────────────────────────────┴───────╯

2025-10-20T04:05:53.302959Z  INFO oarfish::bulk: Total number of alignment records : 272
2025-10-20T04:05:53.302962Z  INFO oarfish::bulk: number of aligned reads : 187
2025-10-20T04:05:53.302965Z  INFO oarfish::bulk: number of unique alignments : 140
2025-10-20T04:05:53.303669Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmppx9ZHJ/file1cf0f926539a7d/config_file_1896697.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Mon Oct 20 00:05:53 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/Rtmppx9ZHJ/file1cf0f926539a7d/sample1_align2genome.bam
sample2 ->/tmp/Rtmppx9ZHJ/file1cf0f926539a7d/sample2_align2genome.bam
sample3 ->/tmp/Rtmppx9ZHJ/file1cf0f926539a7d/sample3_align2genome.bam
-- Running step: isoform_identification @ Mon Oct 20 00:06:11 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Oct 20 00:06:11 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/Rtmppx9ZHJ/file1cf0f926539a7d/sample1_realign2transcript.bam
sample2 ->/tmp/Rtmppx9ZHJ/file1cf0f926539a7d/sample2_realign2transcript.bam
sample3 ->/tmp/Rtmppx9ZHJ/file1cf0f926539a7d/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Mon Oct 20 00:06:30 2025 ----------
2025-10-20T04:06:30.586153Z  INFO oarfish: setting user-provided filter parameters.
2025-10-20T04:06:30.586657Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmppx9ZHJ/file1cf0f926539a7d/sample1_realign2transcript.bam, contains 10 reference sequences.
2025-10-20T04:06:30.586667Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-20T04:06:30.586683Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-20T04:06:30.586754Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-20T04:06:30.586760Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-20T04:06:30.589639Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-20T04:06:30.589781Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 707   │
│ aligned fraction too low        │ 2     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 98    │
╰─────────────────────────────────┴───────╯

2025-10-20T04:06:30.589802Z  INFO oarfish::bulk: Total number of alignment records : 125
2025-10-20T04:06:30.589805Z  INFO oarfish::bulk: number of aligned reads : 98
2025-10-20T04:06:30.589808Z  INFO oarfish::bulk: number of unique alignments : 86
2025-10-20T04:06:30.590470Z  INFO oarfish: oarfish completed successfully.
2025-10-20T04:06:30.602993Z  INFO oarfish: setting user-provided filter parameters.
2025-10-20T04:06:30.603381Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmppx9ZHJ/file1cf0f926539a7d/sample2_realign2transcript.bam, contains 10 reference sequences.
2025-10-20T04:06:30.603393Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-20T04:06:30.603396Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-20T04:06:30.603470Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-20T04:06:30.603477Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-20T04:06:30.606193Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-20T04:06:30.606328Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 701   │
│ aligned fraction too low        │ 3     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 97    │
╰─────────────────────────────────┴───────╯

2025-10-20T04:06:30.606355Z  INFO oarfish::bulk: Total number of alignment records : 136
2025-10-20T04:06:30.606357Z  INFO oarfish::bulk: number of aligned reads : 97
2025-10-20T04:06:30.606369Z  INFO oarfish::bulk: number of unique alignments : 79
2025-10-20T04:06:30.606988Z  INFO oarfish: oarfish completed successfully.
2025-10-20T04:06:30.617723Z  INFO oarfish: setting user-provided filter parameters.
2025-10-20T04:06:30.618099Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmppx9ZHJ/file1cf0f926539a7d/sample3_realign2transcript.bam, contains 10 reference sequences.
2025-10-20T04:06:30.618124Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-20T04:06:30.618132Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-20T04:06:30.618195Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-20T04:06:30.618201Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-20T04:06:30.622539Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-20T04:06:30.622701Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 1060  │
│ aligned fraction too low        │ 6     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 187   │
╰─────────────────────────────────┴───────╯

2025-10-20T04:06:30.622729Z  INFO oarfish::bulk: Total number of alignment records : 272
2025-10-20T04:06:30.622731Z  INFO oarfish::bulk: number of aligned reads : 187
2025-10-20T04:06:30.622734Z  INFO oarfish::bulk: number of unique alignments : 140
2025-10-20T04:06:30.623459Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmppx9ZHJ/file1cf0f96465b786/config_file_1896697.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Mon Oct 20 00:06:30 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/Rtmppx9ZHJ/file1cf0f96465b786/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/Rtmppx9ZHJ/file1cf0f96465b786/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/Rtmppx9ZHJ/file1cf0f96465b786/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Mon Oct 20 00:06:31 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Oct 20 00:06:32 2025 -------------------
Realigning sample sample1 -> /tmp/Rtmppx9ZHJ/file1cf0f96465b786/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/Rtmppx9ZHJ/file1cf0f96465b786/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/Rtmppx9ZHJ/file1cf0f96465b786/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Mon Oct 20 00:06:32 2025 ----------
00:06:32 Mon Oct 20 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmppx9ZHJ/file1cf0f954186078/config_file_1896697.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Mon Oct 20 00:06:33 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/Rtmppx9ZHJ/file1cf0f954186078/sample1_align2genome.bam
sample2 ->/tmp/Rtmppx9ZHJ/file1cf0f954186078/sample2_align2genome.bam
sample3 ->/tmp/Rtmppx9ZHJ/file1cf0f954186078/sample3_align2genome.bam
-- Running step: isoform_identification @ Mon Oct 20 00:06:52 2025 -------------
Inputs:  ['/tmp/Rtmppx9ZHJ/file1cf0f96465b786/sample1_realign2transcript.bam', '/tmp/Rtmppx9ZHJ/file1cf0f96465b786/sample2_realign2transcript.bam', '/tmp/Rtmppx9ZHJ/file1cf0f96465b786/sample3_realign2transcript.bam'] /tmp/Rtmppx9ZHJ/file1cf0f96465b786/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 391, 'not_enough_coverage': 2})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Oct 20 00:06:52 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/Rtmppx9ZHJ/file1cf0f954186078/sample1_realign2transcript.bam
sample2 ->/tmp/Rtmppx9ZHJ/file1cf0f954186078/sample2_realign2transcript.bam
sample3 ->/tmp/Rtmppx9ZHJ/file1cf0f954186078/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Mon Oct 20 00:07:10 2025 ----------
00:07:10 Mon Oct 20 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmppx9ZHJ/file1cf0f943072649/config_file_1896697.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Mon Oct 20 00:07:11 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmppx9ZHJ/file1cf0f943072649/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Mon Oct 20 00:07:11 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmppx9ZHJ/file1cf0f943072649/matched_reads.fastq.gz -> /tmp/Rtmppx9ZHJ/file1cf0f943072649/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Mon Oct 20 00:07:12 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---
Detected 2 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Mon Oct 20 00:07:21 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmppx9ZHJ/file1cf0f943072649/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmppx9ZHJ/file1cf0f943072649/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/Rtmppx9ZHJ/file1cf0f943072649/matched_reads.fastq.gz -> /tmp/Rtmppx9ZHJ/file1cf0f943072649/realign2transcript.bam
Sorting BAM files by 8 with CB threads...

[bam_sort_core] merging from 0 files and 8 in-memory blocks...
-- Running step: transcript_quantification @ Mon Oct 20 00:07:21 2025 ----------
2025-10-20T04:07:21.500740Z  INFO oarfish: setting user-provided filter parameters.
2025-10-20T04:07:21.501169Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmppx9ZHJ/file1cf0f943072649/realign2transcript.bam, contains 5 reference sequences.
2025-10-20T04:07:21.501181Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-20T04:07:21.501185Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-20T04:07:21.501241Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-20T04:07:21.501248Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-20T04:07:21.507769Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmppx9ZHJ/file1cf0f91148e25a/config_file_1896697.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Mon Oct 20 00:07:22 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmppx9ZHJ/file1cf0f91148e25a/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Mon Oct 20 00:07:22 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmppx9ZHJ/file1cf0f91148e25a/matched_reads.fastq.gz ->/tmp/Rtmppx9ZHJ/file1cf0f91148e25a/align2genome.bam
-- Running step: isoform_identification @ Mon Oct 20 00:07:39 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---
Detected 2 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Mon Oct 20 00:07:49 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmppx9ZHJ/file1cf0f91148e25a/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmppx9ZHJ/file1cf0f91148e25a/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/Rtmppx9ZHJ/file1cf0f91148e25a/matched_reads.fastq.gz ->/tmp/Rtmppx9ZHJ/file1cf0f91148e25a/realign2transcript.bam
-- Running step: transcript_quantification @ Mon Oct 20 00:08:06 2025 ----------
2025-10-20T04:08:06.340386Z  INFO oarfish: setting user-provided filter parameters.
2025-10-20T04:08:06.340846Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmppx9ZHJ/file1cf0f91148e25a/realign2transcript.bam, contains 5 reference sequences.
2025-10-20T04:08:06.340856Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-20T04:08:06.340860Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-20T04:08:06.340919Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-20T04:08:06.340926Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-20T04:08:06.347569Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmppx9ZHJ/file1cf0f96a59f1cc/config_file_1896697.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Mon Oct 20 00:08:06 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmppx9ZHJ/file1cf0f96a59f1cc/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Mon Oct 20 00:08:07 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmppx9ZHJ/file1cf0f96a59f1cc/matched_reads.fastq.gz -> /tmp/Rtmppx9ZHJ/file1cf0f96a59f1cc/align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Mon Oct 20 00:08:07 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---
Detected 2 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Mon Oct 20 00:08:16 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmppx9ZHJ/file1cf0f96a59f1cc/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmppx9ZHJ/file1cf0f96a59f1cc/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/Rtmppx9ZHJ/file1cf0f96a59f1cc/matched_reads.fastq.gz -> /tmp/Rtmppx9ZHJ/file1cf0f96a59f1cc/realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 8 threads...

[bam_sort_core] merging from 0 files and 8 in-memory blocks...
Indexing bam files
-- Running step: transcript_quantification @ Mon Oct 20 00:08:16 2025 ----------
00:08:16 Mon Oct 20 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Inputs:  ['/tmp/Rtmppx9ZHJ/file1cf0f954186078/sample1_realign2transcript.bam', '/tmp/Rtmppx9ZHJ/file1cf0f954186078/sample2_realign2transcript.bam', '/tmp/Rtmppx9ZHJ/file1cf0f954186078/sample3_realign2transcript.bam'] /tmp/Rtmppx9ZHJ/file1cf0f954186078/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 391, 'not_enough_coverage': 2})
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmppx9ZHJ/file1cf0f94eafa89b/config_file_1896697.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Mon Oct 20 00:08:17 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmppx9ZHJ/file1cf0f94eafa89b/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Mon Oct 20 00:08:17 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmppx9ZHJ/file1cf0f94eafa89b/matched_reads.fastq.gz ->/tmp/Rtmppx9ZHJ/file1cf0f94eafa89b/align2genome.bam
-- Running step: isoform_identification @ Mon Oct 20 00:08:35 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---
Detected 2 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Mon Oct 20 00:08:44 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmppx9ZHJ/file1cf0f94eafa89b/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmppx9ZHJ/file1cf0f94eafa89b/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/Rtmppx9ZHJ/file1cf0f94eafa89b/matched_reads.fastq.gz ->/tmp/Rtmppx9ZHJ/file1cf0f94eafa89b/realign2transcript.bam
-- Running step: transcript_quantification @ Mon Oct 20 00:09:01 2025 ----------
00:09:01 Mon Oct 20 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
	Counter({'counted_reads': 354, 'not_enough_coverage': 12, 'unmapped': 6})
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmppx9ZHJ/file1cf0f97cb3cf8d/config_file_1896697.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Mon Oct 20 00:09:02 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmppx9ZHJ/file1cf0f97cb3cf8d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Mon Oct 20 00:09:02 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmppx9ZHJ/file1cf0f97cb3cf8d/matched_reads.fastq.gz -> /tmp/Rtmppx9ZHJ/file1cf0f97cb3cf8d/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Mon Oct 20 00:09:02 2025 -------------
	Counter({'counted_reads': 354, 'not_enough_coverage': 12, 'unmapped': 6})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Oct 20 00:09:03 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmppx9ZHJ/file1cf0f97cb3cf8d/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmppx9ZHJ/file1cf0f97cb3cf8d/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/Rtmppx9ZHJ/file1cf0f97cb3cf8d/matched_reads.fastq.gz -> /tmp/Rtmppx9ZHJ/file1cf0f97cb3cf8d/realign2transcript.bam
Sorting BAM files by 8 with CB threads...

[bam_sort_core] merging from 0 files and 8 in-memory blocks...
-- Running step: transcript_quantification @ Mon Oct 20 00:09:03 2025 ----------
2025-10-20T04:09:03.324535Z  INFO oarfish: setting user-provided filter parameters.
2025-10-20T04:09:03.325055Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmppx9ZHJ/file1cf0f97cb3cf8d/realign2transcript.bam, contains 10 reference sequences.
2025-10-20T04:09:03.325064Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-20T04:09:03.325067Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-20T04:09:03.325142Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-20T04:09:03.325153Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-20T04:09:03.335205Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmppx9ZHJ/file1cf0f92fc1e4d4/config_file_1896697.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Mon Oct 20 00:09:04 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmppx9ZHJ/file1cf0f92fc1e4d4/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Mon Oct 20 00:09:04 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmppx9ZHJ/file1cf0f92fc1e4d4/matched_reads.fastq.gz ->/tmp/Rtmppx9ZHJ/file1cf0f92fc1e4d4/align2genome.bam
-- Running step: isoform_identification @ Mon Oct 20 00:09:21 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Oct 20 00:09:21 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmppx9ZHJ/file1cf0f92fc1e4d4/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmppx9ZHJ/file1cf0f92fc1e4d4/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/Rtmppx9ZHJ/file1cf0f92fc1e4d4/matched_reads.fastq.gz ->/tmp/Rtmppx9ZHJ/file1cf0f92fc1e4d4/realign2transcript.bam
-- Running step: transcript_quantification @ Mon Oct 20 00:09:39 2025 ----------
2025-10-20T04:09:39.026388Z  INFO oarfish: setting user-provided filter parameters.
2025-10-20T04:09:39.026974Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmppx9ZHJ/file1cf0f92fc1e4d4/realign2transcript.bam, contains 10 reference sequences.
2025-10-20T04:09:39.026985Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-20T04:09:39.026989Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-20T04:09:39.027066Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-20T04:09:39.027074Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-20T04:09:39.036849Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmppx9ZHJ/file1cf0f970937c49/config_file_1896697.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Mon Oct 20 00:09:39 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmppx9ZHJ/file1cf0f970937c49/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Mon Oct 20 00:09:40 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmppx9ZHJ/file1cf0f970937c49/matched_reads.fastq.gz -> /tmp/Rtmppx9ZHJ/file1cf0f970937c49/align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Mon Oct 20 00:09:40 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Oct 20 00:09:40 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmppx9ZHJ/file1cf0f970937c49/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmppx9ZHJ/file1cf0f970937c49/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/Rtmppx9ZHJ/file1cf0f970937c49/matched_reads.fastq.gz -> /tmp/Rtmppx9ZHJ/file1cf0f970937c49/realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 8 threads...

[bam_sort_core] merging from 0 files and 8 in-memory blocks...
Indexing bam files
-- Running step: transcript_quantification @ Mon Oct 20 00:09:40 2025 ----------
00:09:40 Mon Oct 20 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmppx9ZHJ/file1cf0f9cf14f5f/config_file_1896697.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Mon Oct 20 00:09:41 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmppx9ZHJ/file1cf0f9cf14f5f/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Mon Oct 20 00:09:42 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmppx9ZHJ/file1cf0f9cf14f5f/matched_reads.fastq.gz ->/tmp/Rtmppx9ZHJ/file1cf0f9cf14f5f/align2genome.bam
-- Running step: isoform_identification @ Mon Oct 20 00:10:00 2025 -------------
	Counter({'counted_reads': 368, 'unmapped': 4})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Oct 20 00:10:00 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmppx9ZHJ/file1cf0f9cf14f5f/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmppx9ZHJ/file1cf0f9cf14f5f/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/Rtmppx9ZHJ/file1cf0f9cf14f5f/matched_reads.fastq.gz ->/tmp/Rtmppx9ZHJ/file1cf0f9cf14f5f/realign2transcript.bam
-- Running step: transcript_quantification @ Mon Oct 20 00:10:17 2025 ----------
00:10:17 Mon Oct 20 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmppx9ZHJ/file1cf0f96cc79ea6/config_file_1896697.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Mon Oct 20 00:10:19 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmppx9ZHJ/file1cf0f96cc79ea6/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmppx9ZHJ/file1cf0f96cc79ea6/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmppx9ZHJ/file1cf0f96cc79ea6/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmppx9ZHJ/file1cf0f96cc79ea6/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmppx9ZHJ/file1cf0f96cc79ea6/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmppx9ZHJ/file1cf0f96cc79ea6/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
Reads	Barcodes
4	1
3	9
2	9
1	44
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmppx9ZHJ/file1cf0f96cc79ea6/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmppx9ZHJ/file1cf0f96cc79ea6/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
Reads	Barcodes
4	2
3	3
2	16
1	47
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmppx9ZHJ/file1cf0f96cc79ea6/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmppx9ZHJ/file1cf0f96cc79ea6/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Mon Oct 20 00:10:19 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmppx9ZHJ/file1cf0f96cc79ea6/sampleA_matched_reads.fastq.gz -> /tmp/Rtmppx9ZHJ/file1cf0f96cc79ea6/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmppx9ZHJ/file1cf0f96cc79ea6/sample1_matched_reads.fastq.gz -> /tmp/Rtmppx9ZHJ/file1cf0f96cc79ea6/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmppx9ZHJ/file1cf0f96cc79ea6/sample2_matched_reads.fastq.gz -> /tmp/Rtmppx9ZHJ/file1cf0f96cc79ea6/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmppx9ZHJ/file1cf0f96cc79ea6/sample3_matched_reads.fastq.gz -> /tmp/Rtmppx9ZHJ/file1cf0f96cc79ea6/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Mon Oct 20 00:10:21 2025 ----------------
00:10:21 Mon Oct 20 2025 quantify genes 
Using BAM(s): '/tmp/Rtmppx9ZHJ/file1cf0f96cc79ea6/sampleA_align2genome.bam',
'/tmp/Rtmppx9ZHJ/file1cf0f96cc79ea6/sample1_align2genome.bam',
'/tmp/Rtmppx9ZHJ/file1cf0f96cc79ea6/sample2_align2genome.bam', and
'/tmp/Rtmppx9ZHJ/file1cf0f96cc79ea6/sample3_align2genome.bam'
	Counter({'counted_reads': 368, 'unmapped': 4})
parsing /tmp/Rtmppx9ZHJ/file1cf0f96cc79ea6/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.27gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 402169.29Read/s]
parsing /tmp/Rtmppx9ZHJ/file1cf0f96cc79ea6/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 47.15gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1431894.03Read/s]
parsing /tmp/Rtmppx9ZHJ/file1cf0f96cc79ea6/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 49.86gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1349345.00Read/s]
parsing /tmp/Rtmppx9ZHJ/file1cf0f96cc79ea6/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 35.33gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 774028.20Read/s]
-- Running step: isoform_identification @ Mon Oct 20 00:10:23 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
  |                                                                      |   0%
  |                                                                            
  |==================                                                    |  25%
  |                                                                            
  |===================================                                   |  50%
  |                                                                            
  |====================================================                  |  75%
  |                                                                            
  |======================================================================| 100%

Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Mon Oct 20 00:10:45 2025 -------------------
Checking for fastq file(s) /tmp/Rtmppx9ZHJ/file1cf0f96cc79ea6/fastq, /tmp/Rtmppx9ZHJ/file1cf0f96cc79ea6/fastq/sample1.fq.gz, /tmp/Rtmppx9ZHJ/file1cf0f96cc79ea6/fastq/sample2.fq.gz, /tmp/Rtmppx9ZHJ/file1cf0f96cc79ea6/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmppx9ZHJ/file1cf0f96cc79ea6/sampleA_matched_reads.fastq.gz, /tmp/Rtmppx9ZHJ/file1cf0f96cc79ea6/sample1_matched_reads.fastq.gz, /tmp/Rtmppx9ZHJ/file1cf0f96cc79ea6/sample2_matched_reads.fastq.gz, /tmp/Rtmppx9ZHJ/file1cf0f96cc79ea6/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmppx9ZHJ/file1cf0f96cc79ea6/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmppx9ZHJ/file1cf0f96cc79ea6/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmppx9ZHJ/file1cf0f96cc79ea6/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmppx9ZHJ/file1cf0f96cc79ea6/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmppx9ZHJ/file1cf0f96cc79ea6/sampleA_matched_reads_dedup.fastq.gz -> /tmp/Rtmppx9ZHJ/file1cf0f96cc79ea6/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmppx9ZHJ/file1cf0f96cc79ea6/sample1_matched_reads_dedup.fastq.gz -> /tmp/Rtmppx9ZHJ/file1cf0f96cc79ea6/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmppx9ZHJ/file1cf0f96cc79ea6/sample2_matched_reads_dedup.fastq.gz -> /tmp/Rtmppx9ZHJ/file1cf0f96cc79ea6/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmppx9ZHJ/file1cf0f96cc79ea6/sample3_matched_reads_dedup.fastq.gz -> /tmp/Rtmppx9ZHJ/file1cf0f96cc79ea6/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Mon Oct 20 00:10:46 2025 ----------
2025-10-20T04:10:46.756305Z  INFO oarfish: setting user-provided filter parameters.
2025-10-20T04:10:46.756682Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmppx9ZHJ/file1cf0f96cc79ea6/sampleA_realign2transcript.bam, contains 5 reference sequences.
2025-10-20T04:10:46.756701Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-20T04:10:46.756704Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-20T04:10:46.756755Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-20T04:10:46.756760Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-20T04:10:46.762419Z  INFO oarfish::single_cell: Processed 100 cells.
2025-10-20T04:10:47.054702Z  INFO oarfish: setting user-provided filter parameters.
2025-10-20T04:10:47.055065Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmppx9ZHJ/file1cf0f96cc79ea6/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-10-20T04:10:47.055072Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-20T04:10:47.055075Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-20T04:10:47.055134Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-20T04:10:47.055142Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-20T04:10:47.343164Z  INFO oarfish: setting user-provided filter parameters.
2025-10-20T04:10:47.343521Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmppx9ZHJ/file1cf0f96cc79ea6/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-10-20T04:10:47.343529Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-20T04:10:47.343532Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-20T04:10:47.343584Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-20T04:10:47.343590Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-20T04:10:47.658222Z  INFO oarfish: setting user-provided filter parameters.
2025-10-20T04:10:47.658583Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmppx9ZHJ/file1cf0f96cc79ea6/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-10-20T04:10:47.658590Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-20T04:10:47.658594Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-20T04:10:47.658648Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-20T04:10:47.658654Z  INFO oarfish: parsed reference information for 5 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmppx9ZHJ/file1cf0f96fa09a5a/config_file_1896697.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Mon Oct 20 00:10:48 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmppx9ZHJ/file1cf0f96fa09a5a/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmppx9ZHJ/file1cf0f96fa09a5a/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmppx9ZHJ/file1cf0f96fa09a5a/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmppx9ZHJ/file1cf0f96fa09a5a/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmppx9ZHJ/file1cf0f96fa09a5a/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmppx9ZHJ/file1cf0f96fa09a5a/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
Reads	Barcodes
4	1
3	9
2	9
1	44
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmppx9ZHJ/file1cf0f96fa09a5a/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmppx9ZHJ/file1cf0f96fa09a5a/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
Reads	Barcodes
4	2
3	3
2	16
1	47
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmppx9ZHJ/file1cf0f96fa09a5a/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmppx9ZHJ/file1cf0f96fa09a5a/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Mon Oct 20 00:10:48 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmppx9ZHJ/file1cf0f96fa09a5a/sampleA_matched_reads.fastq.gz ->/tmp/Rtmppx9ZHJ/file1cf0f96fa09a5a/sampleA_align2genome.bam
/tmp/Rtmppx9ZHJ/file1cf0f96fa09a5a/sample1_matched_reads.fastq.gz ->/tmp/Rtmppx9ZHJ/file1cf0f96fa09a5a/sample1_align2genome.bam
/tmp/Rtmppx9ZHJ/file1cf0f96fa09a5a/sample2_matched_reads.fastq.gz ->/tmp/Rtmppx9ZHJ/file1cf0f96fa09a5a/sample2_align2genome.bam
/tmp/Rtmppx9ZHJ/file1cf0f96fa09a5a/sample3_matched_reads.fastq.gz ->/tmp/Rtmppx9ZHJ/file1cf0f96fa09a5a/sample3_align2genome.bam
-- Running step: gene_quantification @ Mon Oct 20 00:11:07 2025 ----------------
00:11:07 Mon Oct 20 2025 quantify genes 
Using BAM(s): '/tmp/Rtmppx9ZHJ/file1cf0f96fa09a5a/sampleA_align2genome.bam',
'/tmp/Rtmppx9ZHJ/file1cf0f96fa09a5a/sample1_align2genome.bam',
'/tmp/Rtmppx9ZHJ/file1cf0f96fa09a5a/sample2_align2genome.bam', and
'/tmp/Rtmppx9ZHJ/file1cf0f96fa09a5a/sample3_align2genome.bam'
parsing /tmp/Rtmppx9ZHJ/file1cf0f96fa09a5a/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.26gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 392592.76Read/s]
parsing /tmp/Rtmppx9ZHJ/file1cf0f96fa09a5a/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.14gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1010870.53Read/s]
parsing /tmp/Rtmppx9ZHJ/file1cf0f96fa09a5a/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 47.59gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1276105.63Read/s]
parsing /tmp/Rtmppx9ZHJ/file1cf0f96fa09a5a/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 33.14gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 742354.69Read/s]
-- Running step: isoform_identification @ Mon Oct 20 00:11:08 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
  |                                                                      |   0%
  |                                                                            
  |==================                                                    |  25%
  |                                                                            
  |===================================                                   |  50%
  |                                                                            
  |====================================================                  |  75%
  |                                                                            
  |======================================================================| 100%

Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Mon Oct 20 00:11:31 2025 -------------------
Checking for fastq file(s) /tmp/Rtmppx9ZHJ/file1cf0f96fa09a5a/fastq, /tmp/Rtmppx9ZHJ/file1cf0f96fa09a5a/fastq/sample1.fq.gz, /tmp/Rtmppx9ZHJ/file1cf0f96fa09a5a/fastq/sample2.fq.gz, /tmp/Rtmppx9ZHJ/file1cf0f96fa09a5a/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmppx9ZHJ/file1cf0f96fa09a5a/sampleA_matched_reads.fastq.gz, /tmp/Rtmppx9ZHJ/file1cf0f96fa09a5a/sample1_matched_reads.fastq.gz, /tmp/Rtmppx9ZHJ/file1cf0f96fa09a5a/sample2_matched_reads.fastq.gz, /tmp/Rtmppx9ZHJ/file1cf0f96fa09a5a/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmppx9ZHJ/file1cf0f96fa09a5a/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmppx9ZHJ/file1cf0f96fa09a5a/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmppx9ZHJ/file1cf0f96fa09a5a/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmppx9ZHJ/file1cf0f96fa09a5a/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/Rtmppx9ZHJ/file1cf0f96fa09a5a/sampleA_matched_reads_dedup.fastq.gz ->/tmp/Rtmppx9ZHJ/file1cf0f96fa09a5a/sampleA_realign2transcript.bam
/tmp/Rtmppx9ZHJ/file1cf0f96fa09a5a/sample1_matched_reads_dedup.fastq.gz ->/tmp/Rtmppx9ZHJ/file1cf0f96fa09a5a/sample1_realign2transcript.bam
/tmp/Rtmppx9ZHJ/file1cf0f96fa09a5a/sample2_matched_reads_dedup.fastq.gz ->/tmp/Rtmppx9ZHJ/file1cf0f96fa09a5a/sample2_realign2transcript.bam
/tmp/Rtmppx9ZHJ/file1cf0f96fa09a5a/sample3_matched_reads_dedup.fastq.gz ->/tmp/Rtmppx9ZHJ/file1cf0f96fa09a5a/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Mon Oct 20 00:11:50 2025 ----------
2025-10-20T04:11:50.663053Z  INFO oarfish: setting user-provided filter parameters.
2025-10-20T04:11:50.663532Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmppx9ZHJ/file1cf0f96fa09a5a/sampleA_realign2transcript.bam, contains 5 reference sequences.
2025-10-20T04:11:50.663543Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-20T04:11:50.663546Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-20T04:11:50.663594Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-20T04:11:50.663600Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-20T04:11:50.669753Z  INFO oarfish::single_cell: Processed 100 cells.
2025-10-20T04:11:51.033575Z  INFO oarfish: setting user-provided filter parameters.
2025-10-20T04:11:51.034111Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmppx9ZHJ/file1cf0f96fa09a5a/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-10-20T04:11:51.034130Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-20T04:11:51.034134Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-20T04:11:51.034190Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-20T04:11:51.034197Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-20T04:11:51.349833Z  INFO oarfish: setting user-provided filter parameters.
2025-10-20T04:11:51.350229Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmppx9ZHJ/file1cf0f96fa09a5a/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-10-20T04:11:51.350241Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-20T04:11:51.350246Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-20T04:11:51.350303Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-20T04:11:51.350309Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-20T04:11:51.675537Z  INFO oarfish: setting user-provided filter parameters.
2025-10-20T04:11:51.676052Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmppx9ZHJ/file1cf0f96fa09a5a/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-10-20T04:11:51.676063Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-20T04:11:51.676066Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-20T04:11:51.676130Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-20T04:11:51.676147Z  INFO oarfish: parsed reference information for 5 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmppx9ZHJ/file1cf0f914e2a73/config_file_1896697.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Mon Oct 20 00:11:52 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmppx9ZHJ/file1cf0f914e2a73/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmppx9ZHJ/file1cf0f914e2a73/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmppx9ZHJ/file1cf0f914e2a73/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmppx9ZHJ/file1cf0f914e2a73/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmppx9ZHJ/file1cf0f914e2a73/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmppx9ZHJ/file1cf0f914e2a73/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
Reads	Barcodes
4	1
3	9
2	9
1	44
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmppx9ZHJ/file1cf0f914e2a73/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmppx9ZHJ/file1cf0f914e2a73/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
Reads	Barcodes
4	2
3	3
2	16
1	47
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmppx9ZHJ/file1cf0f914e2a73/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmppx9ZHJ/file1cf0f914e2a73/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Mon Oct 20 00:11:52 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmppx9ZHJ/file1cf0f914e2a73/sampleA_matched_reads.fastq.gz -> /tmp/Rtmppx9ZHJ/file1cf0f914e2a73/sampleA_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmppx9ZHJ/file1cf0f914e2a73/sample1_matched_reads.fastq.gz -> /tmp/Rtmppx9ZHJ/file1cf0f914e2a73/sample1_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmppx9ZHJ/file1cf0f914e2a73/sample2_matched_reads.fastq.gz -> /tmp/Rtmppx9ZHJ/file1cf0f914e2a73/sample2_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmppx9ZHJ/file1cf0f914e2a73/sample3_matched_reads.fastq.gz -> /tmp/Rtmppx9ZHJ/file1cf0f914e2a73/sample3_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Mon Oct 20 00:11:54 2025 ----------------
00:11:54 Mon Oct 20 2025 quantify genes 
Using BAM(s): '/tmp/Rtmppx9ZHJ/file1cf0f914e2a73/sampleA_align2genome.bam',
'/tmp/Rtmppx9ZHJ/file1cf0f914e2a73/sample1_align2genome.bam',
'/tmp/Rtmppx9ZHJ/file1cf0f914e2a73/sample2_align2genome.bam', and
'/tmp/Rtmppx9ZHJ/file1cf0f914e2a73/sample3_align2genome.bam'
parsing /tmp/Rtmppx9ZHJ/file1cf0f914e2a73/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.08gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 422829.95Read/s]
parsing /tmp/Rtmppx9ZHJ/file1cf0f914e2a73/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.59gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1391976.64Read/s]
parsing /tmp/Rtmppx9ZHJ/file1cf0f914e2a73/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 48.34gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1289127.12Read/s]
parsing /tmp/Rtmppx9ZHJ/file1cf0f914e2a73/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 34.25gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 777529.29Read/s]
-- Running step: isoform_identification @ Mon Oct 20 00:11:55 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
  |                                                                      |   0%
  |                                                                            
  |==================                                                    |  25%
  |                                                                            
  |===================================                                   |  50%
  |                                                                            
  |====================================================                  |  75%
  |                                                                            
  |======================================================================| 100%

Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Mon Oct 20 00:12:18 2025 -------------------
Checking for fastq file(s) /tmp/Rtmppx9ZHJ/file1cf0f914e2a73/fastq, /tmp/Rtmppx9ZHJ/file1cf0f914e2a73/fastq/sample1.fq.gz, /tmp/Rtmppx9ZHJ/file1cf0f914e2a73/fastq/sample2.fq.gz, /tmp/Rtmppx9ZHJ/file1cf0f914e2a73/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmppx9ZHJ/file1cf0f914e2a73/sampleA_matched_reads.fastq.gz, /tmp/Rtmppx9ZHJ/file1cf0f914e2a73/sample1_matched_reads.fastq.gz, /tmp/Rtmppx9ZHJ/file1cf0f914e2a73/sample2_matched_reads.fastq.gz, /tmp/Rtmppx9ZHJ/file1cf0f914e2a73/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmppx9ZHJ/file1cf0f914e2a73/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmppx9ZHJ/file1cf0f914e2a73/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmppx9ZHJ/file1cf0f914e2a73/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmppx9ZHJ/file1cf0f914e2a73/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmppx9ZHJ/file1cf0f914e2a73/sampleA_matched_reads_dedup.fastq.gz -> /tmp/Rtmppx9ZHJ/file1cf0f914e2a73/sampleA_realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample /tmp/Rtmppx9ZHJ/file1cf0f914e2a73/sample1_matched_reads_dedup.fastq.gz -> /tmp/Rtmppx9ZHJ/file1cf0f914e2a73/sample1_realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample /tmp/Rtmppx9ZHJ/file1cf0f914e2a73/sample2_matched_reads_dedup.fastq.gz -> /tmp/Rtmppx9ZHJ/file1cf0f914e2a73/sample2_realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample /tmp/Rtmppx9ZHJ/file1cf0f914e2a73/sample3_matched_reads_dedup.fastq.gz -> /tmp/Rtmppx9ZHJ/file1cf0f914e2a73/sample3_realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Mon Oct 20 00:12:19 2025 ----------
00:12:19 Mon Oct 20 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/Rtmppx9ZHJ/file1cf0f914e2a73/sampleA_realign2transcript.bam...
parsing /tmp/Rtmppx9ZHJ/file1cf0f914e2a73/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmppx9ZHJ/file1cf0f914e2a73/sampleA_realign2transcript.bamdone
parsing /tmp/Rtmppx9ZHJ/file1cf0f914e2a73/sample1_realign2transcript.bam...
parsing /tmp/Rtmppx9ZHJ/file1cf0f914e2a73/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmppx9ZHJ/file1cf0f914e2a73/sample1_realign2transcript.bamdone
parsing /tmp/Rtmppx9ZHJ/file1cf0f914e2a73/sample2_realign2transcript.bam...
parsing /tmp/Rtmppx9ZHJ/file1cf0f914e2a73/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmppx9ZHJ/file1cf0f914e2a73/sample2_realign2transcript.bamdone
parsing /tmp/Rtmppx9ZHJ/file1cf0f914e2a73/sample3_realign2transcript.bam...
parsing /tmp/Rtmppx9ZHJ/file1cf0f914e2a73/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmppx9ZHJ/file1cf0f914e2a73/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmppx9ZHJ/file1cf0f924bc2494/config_file_1896697.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Mon Oct 20 00:12:21 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmppx9ZHJ/file1cf0f924bc2494/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmppx9ZHJ/file1cf0f924bc2494/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmppx9ZHJ/file1cf0f924bc2494/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmppx9ZHJ/file1cf0f924bc2494/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmppx9ZHJ/file1cf0f924bc2494/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmppx9ZHJ/file1cf0f924bc2494/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
Reads	Barcodes
4	1
3	9
2	9
1	44
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmppx9ZHJ/file1cf0f924bc2494/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmppx9ZHJ/file1cf0f924bc2494/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
Reads	Barcodes
4	2
3	3
2	16
1	47
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmppx9ZHJ/file1cf0f924bc2494/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmppx9ZHJ/file1cf0f924bc2494/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Mon Oct 20 00:12:22 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmppx9ZHJ/file1cf0f924bc2494/sampleA_matched_reads.fastq.gz ->/tmp/Rtmppx9ZHJ/file1cf0f924bc2494/sampleA_align2genome.bam
/tmp/Rtmppx9ZHJ/file1cf0f924bc2494/sample1_matched_reads.fastq.gz ->/tmp/Rtmppx9ZHJ/file1cf0f924bc2494/sample1_align2genome.bam
/tmp/Rtmppx9ZHJ/file1cf0f924bc2494/sample2_matched_reads.fastq.gz ->/tmp/Rtmppx9ZHJ/file1cf0f924bc2494/sample2_align2genome.bam
/tmp/Rtmppx9ZHJ/file1cf0f924bc2494/sample3_matched_reads.fastq.gz ->/tmp/Rtmppx9ZHJ/file1cf0f924bc2494/sample3_align2genome.bam
-- Running step: gene_quantification @ Mon Oct 20 00:12:41 2025 ----------------
00:12:41 Mon Oct 20 2025 quantify genes 
Using BAM(s): '/tmp/Rtmppx9ZHJ/file1cf0f924bc2494/sampleA_align2genome.bam',
'/tmp/Rtmppx9ZHJ/file1cf0f924bc2494/sample1_align2genome.bam',
'/tmp/Rtmppx9ZHJ/file1cf0f924bc2494/sample2_align2genome.bam', and
'/tmp/Rtmppx9ZHJ/file1cf0f924bc2494/sample3_align2genome.bam'
	Counter({'counted_reads': 347, 'not_enough_coverage': 9, 'unmapped': 2})
	Counter({'counted_reads': 89, 'not_enough_coverage': 2})
	Counter({'counted_reads': 92, 'not_enough_coverage': 3})
	Counter({'counted_reads': 168, 'not_enough_coverage': 6, 'unmapped': 2})
parsing /tmp/Rtmppx9ZHJ/file1cf0f924bc2494/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.65gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 376549.00Read/s]
parsing /tmp/Rtmppx9ZHJ/file1cf0f924bc2494/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 40.15gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1268848.02Read/s]
parsing /tmp/Rtmppx9ZHJ/file1cf0f924bc2494/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 46.76gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1227553.27Read/s]
parsing /tmp/Rtmppx9ZHJ/file1cf0f924bc2494/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 34.32gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 758902.80Read/s]
-- Running step: isoform_identification @ Mon Oct 20 00:12:42 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
  |                                                                      |   0%
  |                                                                            
  |==================                                                    |  25%
  |                                                                            
  |===================================                                   |  50%
  |                                                                            
  |====================================================                  |  75%
  |                                                                            
  |======================================================================| 100%

Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Mon Oct 20 00:13:04 2025 -------------------
Checking for fastq file(s) /tmp/Rtmppx9ZHJ/file1cf0f924bc2494/fastq, /tmp/Rtmppx9ZHJ/file1cf0f924bc2494/fastq/sample1.fq.gz, /tmp/Rtmppx9ZHJ/file1cf0f924bc2494/fastq/sample2.fq.gz, /tmp/Rtmppx9ZHJ/file1cf0f924bc2494/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmppx9ZHJ/file1cf0f924bc2494/sampleA_matched_reads.fastq.gz, /tmp/Rtmppx9ZHJ/file1cf0f924bc2494/sample1_matched_reads.fastq.gz, /tmp/Rtmppx9ZHJ/file1cf0f924bc2494/sample2_matched_reads.fastq.gz, /tmp/Rtmppx9ZHJ/file1cf0f924bc2494/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmppx9ZHJ/file1cf0f924bc2494/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmppx9ZHJ/file1cf0f924bc2494/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmppx9ZHJ/file1cf0f924bc2494/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmppx9ZHJ/file1cf0f924bc2494/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/Rtmppx9ZHJ/file1cf0f924bc2494/sampleA_matched_reads_dedup.fastq.gz ->/tmp/Rtmppx9ZHJ/file1cf0f924bc2494/sampleA_realign2transcript.bam
/tmp/Rtmppx9ZHJ/file1cf0f924bc2494/sample1_matched_reads_dedup.fastq.gz ->/tmp/Rtmppx9ZHJ/file1cf0f924bc2494/sample1_realign2transcript.bam
/tmp/Rtmppx9ZHJ/file1cf0f924bc2494/sample2_matched_reads_dedup.fastq.gz ->/tmp/Rtmppx9ZHJ/file1cf0f924bc2494/sample2_realign2transcript.bam
/tmp/Rtmppx9ZHJ/file1cf0f924bc2494/sample3_matched_reads_dedup.fastq.gz ->/tmp/Rtmppx9ZHJ/file1cf0f924bc2494/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Mon Oct 20 00:13:21 2025 ----------
00:13:21 Mon Oct 20 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/Rtmppx9ZHJ/file1cf0f924bc2494/sampleA_realign2transcript.bam...
parsing /tmp/Rtmppx9ZHJ/file1cf0f924bc2494/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmppx9ZHJ/file1cf0f924bc2494/sampleA_realign2transcript.bamdone
parsing /tmp/Rtmppx9ZHJ/file1cf0f924bc2494/sample1_realign2transcript.bam...
parsing /tmp/Rtmppx9ZHJ/file1cf0f924bc2494/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmppx9ZHJ/file1cf0f924bc2494/sample1_realign2transcript.bamdone
parsing /tmp/Rtmppx9ZHJ/file1cf0f924bc2494/sample2_realign2transcript.bam...
parsing /tmp/Rtmppx9ZHJ/file1cf0f924bc2494/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmppx9ZHJ/file1cf0f924bc2494/sample2_realign2transcript.bamdone
parsing /tmp/Rtmppx9ZHJ/file1cf0f924bc2494/sample3_realign2transcript.bam...
parsing /tmp/Rtmppx9ZHJ/file1cf0f924bc2494/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmppx9ZHJ/file1cf0f924bc2494/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmppx9ZHJ/file1cf0f975001279/config_file_1896697.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Mon Oct 20 00:13:23 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmppx9ZHJ/file1cf0f975001279/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmppx9ZHJ/file1cf0f975001279/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmppx9ZHJ/file1cf0f975001279/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmppx9ZHJ/file1cf0f975001279/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmppx9ZHJ/file1cf0f975001279/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmppx9ZHJ/file1cf0f975001279/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
Reads	Barcodes
4	1
3	9
2	9
1	44
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmppx9ZHJ/file1cf0f975001279/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmppx9ZHJ/file1cf0f975001279/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
Reads	Barcodes
4	2
3	3
2	16
1	47
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmppx9ZHJ/file1cf0f975001279/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmppx9ZHJ/file1cf0f975001279/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Mon Oct 20 00:13:24 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmppx9ZHJ/file1cf0f975001279/sampleA_matched_reads.fastq.gz -> /tmp/Rtmppx9ZHJ/file1cf0f975001279/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmppx9ZHJ/file1cf0f975001279/sample1_matched_reads.fastq.gz -> /tmp/Rtmppx9ZHJ/file1cf0f975001279/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmppx9ZHJ/file1cf0f975001279/sample2_matched_reads.fastq.gz -> /tmp/Rtmppx9ZHJ/file1cf0f975001279/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmppx9ZHJ/file1cf0f975001279/sample3_matched_reads.fastq.gz -> /tmp/Rtmppx9ZHJ/file1cf0f975001279/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Mon Oct 20 00:13:26 2025 ----------------
00:13:26 Mon Oct 20 2025 quantify genes 
Using BAM(s): '/tmp/Rtmppx9ZHJ/file1cf0f975001279/sampleA_align2genome.bam',
'/tmp/Rtmppx9ZHJ/file1cf0f975001279/sample1_align2genome.bam',
'/tmp/Rtmppx9ZHJ/file1cf0f975001279/sample2_align2genome.bam', and
'/tmp/Rtmppx9ZHJ/file1cf0f975001279/sample3_align2genome.bam'
	Counter({'counted_reads': 347, 'not_enough_coverage': 9, 'unmapped': 2})
	Counter({'counted_reads': 89, 'not_enough_coverage': 2})
	Counter({'counted_reads': 92, 'not_enough_coverage': 3})
	Counter({'counted_reads': 168, 'not_enough_coverage': 6, 'unmapped': 2})
parsing /tmp/Rtmppx9ZHJ/file1cf0f975001279/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.92gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 420743.12Read/s]
parsing /tmp/Rtmppx9ZHJ/file1cf0f975001279/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 42.79gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1192919.23Read/s]
parsing /tmp/Rtmppx9ZHJ/file1cf0f975001279/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 50.54gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1339177.52Read/s]
parsing /tmp/Rtmppx9ZHJ/file1cf0f975001279/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 35.55gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 715263.30Read/s]
-- Running step: isoform_identification @ Mon Oct 20 00:13:26 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Oct 20 00:13:27 2025 -------------------
Checking for fastq file(s) /tmp/Rtmppx9ZHJ/file1cf0f975001279/fastq, /tmp/Rtmppx9ZHJ/file1cf0f975001279/fastq/sample1.fq.gz, /tmp/Rtmppx9ZHJ/file1cf0f975001279/fastq/sample2.fq.gz, /tmp/Rtmppx9ZHJ/file1cf0f975001279/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmppx9ZHJ/file1cf0f975001279/sampleA_matched_reads.fastq.gz, /tmp/Rtmppx9ZHJ/file1cf0f975001279/sample1_matched_reads.fastq.gz, /tmp/Rtmppx9ZHJ/file1cf0f975001279/sample2_matched_reads.fastq.gz, /tmp/Rtmppx9ZHJ/file1cf0f975001279/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmppx9ZHJ/file1cf0f975001279/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmppx9ZHJ/file1cf0f975001279/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmppx9ZHJ/file1cf0f975001279/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmppx9ZHJ/file1cf0f975001279/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmppx9ZHJ/file1cf0f975001279/sampleA_matched_reads_dedup.fastq.gz -> /tmp/Rtmppx9ZHJ/file1cf0f975001279/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmppx9ZHJ/file1cf0f975001279/sample1_matched_reads_dedup.fastq.gz -> /tmp/Rtmppx9ZHJ/file1cf0f975001279/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmppx9ZHJ/file1cf0f975001279/sample2_matched_reads_dedup.fastq.gz -> /tmp/Rtmppx9ZHJ/file1cf0f975001279/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmppx9ZHJ/file1cf0f975001279/sample3_matched_reads_dedup.fastq.gz -> /tmp/Rtmppx9ZHJ/file1cf0f975001279/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Mon Oct 20 00:13:29 2025 ----------
2025-10-20T04:13:29.248540Z  INFO oarfish: setting user-provided filter parameters.
2025-10-20T04:13:29.249042Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmppx9ZHJ/file1cf0f975001279/sampleA_realign2transcript.bam, contains 14 reference sequences.
2025-10-20T04:13:29.249050Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-20T04:13:29.249053Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-20T04:13:29.249146Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-20T04:13:29.249157Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-20T04:13:29.261192Z  INFO oarfish::single_cell: Processed 100 cells.
2025-10-20T04:13:29.814921Z  INFO oarfish: setting user-provided filter parameters.
2025-10-20T04:13:29.815523Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmppx9ZHJ/file1cf0f975001279/sample1_realign2transcript.bam, contains 14 reference sequences.
2025-10-20T04:13:29.815536Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-20T04:13:29.815539Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-20T04:13:29.815616Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-20T04:13:29.815624Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-20T04:13:30.356197Z  INFO oarfish: setting user-provided filter parameters.
2025-10-20T04:13:30.356733Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmppx9ZHJ/file1cf0f975001279/sample2_realign2transcript.bam, contains 14 reference sequences.
2025-10-20T04:13:30.356741Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-20T04:13:30.356744Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-20T04:13:30.356819Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-20T04:13:30.356826Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-20T04:13:30.914522Z  INFO oarfish: setting user-provided filter parameters.
2025-10-20T04:13:30.914880Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmppx9ZHJ/file1cf0f975001279/sample3_realign2transcript.bam, contains 14 reference sequences.
2025-10-20T04:13:30.914888Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-20T04:13:30.914891Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-20T04:13:30.914961Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-20T04:13:30.914968Z  INFO oarfish: parsed reference information for 14 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmppx9ZHJ/file1cf0f93e533ce9/config_file_1896697.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Mon Oct 20 00:13:31 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmppx9ZHJ/file1cf0f93e533ce9/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmppx9ZHJ/file1cf0f93e533ce9/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmppx9ZHJ/file1cf0f93e533ce9/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmppx9ZHJ/file1cf0f93e533ce9/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmppx9ZHJ/file1cf0f93e533ce9/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmppx9ZHJ/file1cf0f93e533ce9/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
Reads	Barcodes
4	1
3	9
2	9
1	44
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmppx9ZHJ/file1cf0f93e533ce9/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmppx9ZHJ/file1cf0f93e533ce9/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
Reads	Barcodes
4	2
3	3
2	16
1	47
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmppx9ZHJ/file1cf0f93e533ce9/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmppx9ZHJ/file1cf0f93e533ce9/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Mon Oct 20 00:13:32 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmppx9ZHJ/file1cf0f93e533ce9/sampleA_matched_reads.fastq.gz ->/tmp/Rtmppx9ZHJ/file1cf0f93e533ce9/sampleA_align2genome.bam
/tmp/Rtmppx9ZHJ/file1cf0f93e533ce9/sample1_matched_reads.fastq.gz ->/tmp/Rtmppx9ZHJ/file1cf0f93e533ce9/sample1_align2genome.bam
/tmp/Rtmppx9ZHJ/file1cf0f93e533ce9/sample2_matched_reads.fastq.gz ->/tmp/Rtmppx9ZHJ/file1cf0f93e533ce9/sample2_align2genome.bam
/tmp/Rtmppx9ZHJ/file1cf0f93e533ce9/sample3_matched_reads.fastq.gz ->/tmp/Rtmppx9ZHJ/file1cf0f93e533ce9/sample3_align2genome.bam
-- Running step: gene_quantification @ Mon Oct 20 00:13:50 2025 ----------------
00:13:50 Mon Oct 20 2025 quantify genes 
Using BAM(s): '/tmp/Rtmppx9ZHJ/file1cf0f93e533ce9/sampleA_align2genome.bam',
'/tmp/Rtmppx9ZHJ/file1cf0f93e533ce9/sample1_align2genome.bam',
'/tmp/Rtmppx9ZHJ/file1cf0f93e533ce9/sample2_align2genome.bam', and
'/tmp/Rtmppx9ZHJ/file1cf0f93e533ce9/sample3_align2genome.bam'
parsing /tmp/Rtmppx9ZHJ/file1cf0f93e533ce9/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.58gene_group/s]
/home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/python/count_gene.py:705: RuntimeWarning: More than 20 figures have been opened. Figures created through the pyplot interface (`matplotlib.pyplot.figure`) are retained until explicitly closed and may consume too much memory. (To control this warning, see the rcParam `figure.max_open_warning`). Consider using `matplotlib.pyplot.close()`.
  plt.figure(figsize=(8, 6))
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 388059.66Read/s]
parsing /tmp/Rtmppx9ZHJ/file1cf0f93e533ce9/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.02gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1359844.38Read/s]
parsing /tmp/Rtmppx9ZHJ/file1cf0f93e533ce9/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 49.90gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1311375.69Read/s]
parsing /tmp/Rtmppx9ZHJ/file1cf0f93e533ce9/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 34.91gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 705779.09Read/s]
-- Running step: isoform_identification @ Mon Oct 20 00:13:51 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Oct 20 00:13:52 2025 -------------------
Checking for fastq file(s) /tmp/Rtmppx9ZHJ/file1cf0f93e533ce9/fastq, /tmp/Rtmppx9ZHJ/file1cf0f93e533ce9/fastq/sample1.fq.gz, /tmp/Rtmppx9ZHJ/file1cf0f93e533ce9/fastq/sample2.fq.gz, /tmp/Rtmppx9ZHJ/file1cf0f93e533ce9/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmppx9ZHJ/file1cf0f93e533ce9/sampleA_matched_reads.fastq.gz, /tmp/Rtmppx9ZHJ/file1cf0f93e533ce9/sample1_matched_reads.fastq.gz, /tmp/Rtmppx9ZHJ/file1cf0f93e533ce9/sample2_matched_reads.fastq.gz, /tmp/Rtmppx9ZHJ/file1cf0f93e533ce9/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmppx9ZHJ/file1cf0f93e533ce9/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmppx9ZHJ/file1cf0f93e533ce9/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmppx9ZHJ/file1cf0f93e533ce9/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmppx9ZHJ/file1cf0f93e533ce9/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/Rtmppx9ZHJ/file1cf0f93e533ce9/sampleA_matched_reads_dedup.fastq.gz ->/tmp/Rtmppx9ZHJ/file1cf0f93e533ce9/sampleA_realign2transcript.bam
/tmp/Rtmppx9ZHJ/file1cf0f93e533ce9/sample1_matched_reads_dedup.fastq.gz ->/tmp/Rtmppx9ZHJ/file1cf0f93e533ce9/sample1_realign2transcript.bam
/tmp/Rtmppx9ZHJ/file1cf0f93e533ce9/sample2_matched_reads_dedup.fastq.gz ->/tmp/Rtmppx9ZHJ/file1cf0f93e533ce9/sample2_realign2transcript.bam
/tmp/Rtmppx9ZHJ/file1cf0f93e533ce9/sample3_matched_reads_dedup.fastq.gz ->/tmp/Rtmppx9ZHJ/file1cf0f93e533ce9/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Mon Oct 20 00:14:10 2025 ----------
2025-10-20T04:14:10.829607Z  INFO oarfish: setting user-provided filter parameters.
2025-10-20T04:14:10.830194Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmppx9ZHJ/file1cf0f93e533ce9/sampleA_realign2transcript.bam, contains 14 reference sequences.
2025-10-20T04:14:10.830229Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-20T04:14:10.830234Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-20T04:14:10.830367Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-20T04:14:10.830375Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-20T04:14:10.842305Z  INFO oarfish::single_cell: Processed 100 cells.
2025-10-20T04:14:11.506671Z  INFO oarfish: setting user-provided filter parameters.
2025-10-20T04:14:11.507141Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmppx9ZHJ/file1cf0f93e533ce9/sample1_realign2transcript.bam, contains 14 reference sequences.
2025-10-20T04:14:11.507154Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-20T04:14:11.507158Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-20T04:14:11.507240Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-20T04:14:11.507248Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-20T04:14:12.178517Z  INFO oarfish: setting user-provided filter parameters.
2025-10-20T04:14:12.179196Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmppx9ZHJ/file1cf0f93e533ce9/sample2_realign2transcript.bam, contains 14 reference sequences.
2025-10-20T04:14:12.179209Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-20T04:14:12.179213Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-20T04:14:12.179294Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-20T04:14:12.179303Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-20T04:14:12.710421Z  INFO oarfish: setting user-provided filter parameters.
2025-10-20T04:14:12.710880Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmppx9ZHJ/file1cf0f93e533ce9/sample3_realign2transcript.bam, contains 14 reference sequences.
2025-10-20T04:14:12.710891Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-20T04:14:12.710895Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-20T04:14:12.710974Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-20T04:14:12.710983Z  INFO oarfish: parsed reference information for 14 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/config_file_1896697.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Mon Oct 20 00:14:13 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
Reads	Barcodes
4	1
3	9
2	9
1	44
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
Reads	Barcodes
4	2
3	3
2	16
1	47
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Mon Oct 20 00:14:14 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/sampleA_matched_reads.fastq.gz -> /tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/sampleA_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/sample1_matched_reads.fastq.gz -> /tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/sample1_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/sample2_matched_reads.fastq.gz -> /tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/sample2_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/sample3_matched_reads.fastq.gz -> /tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/sample3_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Mon Oct 20 00:14:15 2025 ----------------
00:14:15 Mon Oct 20 2025 quantify genes 
Using BAM(s): '/tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/sampleA_align2genome.bam',
'/tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/sample1_align2genome.bam',
'/tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/sample2_align2genome.bam', and
'/tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/sample3_align2genome.bam'
parsing /tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.10gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 379602.51Read/s]
parsing /tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  9.44gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  9.42gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1453126.39Read/s]
parsing /tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.98gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1239598.06Read/s]
parsing /tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.60gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 781527.91Read/s]
-- Running step: isoform_identification @ Mon Oct 20 00:14:16 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Oct 20 00:14:17 2025 -------------------
Checking for fastq file(s) /tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/fastq, /tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/fastq/sample1.fq.gz, /tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/fastq/sample2.fq.gz, /tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/sampleA_matched_reads.fastq.gz, /tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/sample1_matched_reads.fastq.gz, /tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/sample2_matched_reads.fastq.gz, /tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/sampleA_matched_reads_dedup.fastq.gz -> /tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/sampleA_realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample /tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/sample1_matched_reads_dedup.fastq.gz -> /tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/sample1_realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample /tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/sample2_matched_reads_dedup.fastq.gz -> /tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/sample2_realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample /tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/sample3_matched_reads_dedup.fastq.gz -> /tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/sample3_realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Mon Oct 20 00:14:18 2025 ----------
00:14:18 Mon Oct 20 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/sampleA_realign2transcript.bam...
parsing /tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/sampleA_realign2transcript.bamdone
parsing /tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/sample1_realign2transcript.bam...
parsing /tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/sample1_realign2transcript.bamdone
parsing /tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/sample2_realign2transcript.bam...
parsing /tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/sample2_realign2transcript.bamdone
parsing /tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/sample3_realign2transcript.bam...
parsing /tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmppx9ZHJ/file1cf0f955f18f3b/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmppx9ZHJ/file1cf0f943cf8560/config_file_1896697.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Mon Oct 20 00:14:21 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmppx9ZHJ/file1cf0f943cf8560/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmppx9ZHJ/file1cf0f943cf8560/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmppx9ZHJ/file1cf0f943cf8560/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmppx9ZHJ/file1cf0f943cf8560/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmppx9ZHJ/file1cf0f943cf8560/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmppx9ZHJ/file1cf0f943cf8560/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
Reads	Barcodes
4	1
3	9
2	9
1	44
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmppx9ZHJ/file1cf0f943cf8560/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmppx9ZHJ/file1cf0f943cf8560/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
Reads	Barcodes
4	2
3	3
2	16
1	47
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmppx9ZHJ/file1cf0f943cf8560/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmppx9ZHJ/file1cf0f943cf8560/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Mon Oct 20 00:14:21 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmppx9ZHJ/file1cf0f943cf8560/sampleA_matched_reads.fastq.gz ->/tmp/Rtmppx9ZHJ/file1cf0f943cf8560/sampleA_align2genome.bam
/tmp/Rtmppx9ZHJ/file1cf0f943cf8560/sample1_matched_reads.fastq.gz ->/tmp/Rtmppx9ZHJ/file1cf0f943cf8560/sample1_align2genome.bam
/tmp/Rtmppx9ZHJ/file1cf0f943cf8560/sample2_matched_reads.fastq.gz ->/tmp/Rtmppx9ZHJ/file1cf0f943cf8560/sample2_align2genome.bam
/tmp/Rtmppx9ZHJ/file1cf0f943cf8560/sample3_matched_reads.fastq.gz ->/tmp/Rtmppx9ZHJ/file1cf0f943cf8560/sample3_align2genome.bam
-- Running step: gene_quantification @ Mon Oct 20 00:14:40 2025 ----------------
00:14:40 Mon Oct 20 2025 quantify genes 
Using BAM(s): '/tmp/Rtmppx9ZHJ/file1cf0f943cf8560/sampleA_align2genome.bam',
'/tmp/Rtmppx9ZHJ/file1cf0f943cf8560/sample1_align2genome.bam',
'/tmp/Rtmppx9ZHJ/file1cf0f943cf8560/sample2_align2genome.bam', and
'/tmp/Rtmppx9ZHJ/file1cf0f943cf8560/sample3_align2genome.bam'
	Counter({'counted_reads': 358})
	Counter({'counted_reads': 91})
	Counter({'counted_reads': 95})
	Counter({'counted_reads': 176})
parsing /tmp/Rtmppx9ZHJ/file1cf0f943cf8560/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 16.60gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 314048.34Read/s]
parsing /tmp/Rtmppx9ZHJ/file1cf0f943cf8560/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 48.56gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1360903.31Read/s]
parsing /tmp/Rtmppx9ZHJ/file1cf0f943cf8560/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 49.86gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1154183.82Read/s]
parsing /tmp/Rtmppx9ZHJ/file1cf0f943cf8560/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 35.71gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 691627.20Read/s]
-- Running step: isoform_identification @ Mon Oct 20 00:14:41 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Oct 20 00:14:41 2025 -------------------
Checking for fastq file(s) /tmp/Rtmppx9ZHJ/file1cf0f943cf8560/fastq, /tmp/Rtmppx9ZHJ/file1cf0f943cf8560/fastq/sample1.fq.gz, /tmp/Rtmppx9ZHJ/file1cf0f943cf8560/fastq/sample2.fq.gz, /tmp/Rtmppx9ZHJ/file1cf0f943cf8560/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmppx9ZHJ/file1cf0f943cf8560/sampleA_matched_reads.fastq.gz, /tmp/Rtmppx9ZHJ/file1cf0f943cf8560/sample1_matched_reads.fastq.gz, /tmp/Rtmppx9ZHJ/file1cf0f943cf8560/sample2_matched_reads.fastq.gz, /tmp/Rtmppx9ZHJ/file1cf0f943cf8560/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmppx9ZHJ/file1cf0f943cf8560/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmppx9ZHJ/file1cf0f943cf8560/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmppx9ZHJ/file1cf0f943cf8560/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmppx9ZHJ/file1cf0f943cf8560/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/Rtmppx9ZHJ/file1cf0f943cf8560/sampleA_matched_reads_dedup.fastq.gz ->/tmp/Rtmppx9ZHJ/file1cf0f943cf8560/sampleA_realign2transcript.bam
/tmp/Rtmppx9ZHJ/file1cf0f943cf8560/sample1_matched_reads_dedup.fastq.gz ->/tmp/Rtmppx9ZHJ/file1cf0f943cf8560/sample1_realign2transcript.bam
/tmp/Rtmppx9ZHJ/file1cf0f943cf8560/sample2_matched_reads_dedup.fastq.gz ->/tmp/Rtmppx9ZHJ/file1cf0f943cf8560/sample2_realign2transcript.bam
/tmp/Rtmppx9ZHJ/file1cf0f943cf8560/sample3_matched_reads_dedup.fastq.gz ->/tmp/Rtmppx9ZHJ/file1cf0f943cf8560/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Mon Oct 20 00:15:00 2025 ----------
00:15:00 Mon Oct 20 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/Rtmppx9ZHJ/file1cf0f943cf8560/sampleA_realign2transcript.bam...
parsing /tmp/Rtmppx9ZHJ/file1cf0f943cf8560/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmppx9ZHJ/file1cf0f943cf8560/sampleA_realign2transcript.bamdone
parsing /tmp/Rtmppx9ZHJ/file1cf0f943cf8560/sample1_realign2transcript.bam...
parsing /tmp/Rtmppx9ZHJ/file1cf0f943cf8560/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmppx9ZHJ/file1cf0f943cf8560/sample1_realign2transcript.bamdone
parsing /tmp/Rtmppx9ZHJ/file1cf0f943cf8560/sample2_realign2transcript.bam...
parsing /tmp/Rtmppx9ZHJ/file1cf0f943cf8560/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmppx9ZHJ/file1cf0f943cf8560/sample2_realign2transcript.bamdone
parsing /tmp/Rtmppx9ZHJ/file1cf0f943cf8560/sample3_realign2transcript.bam...
parsing /tmp/Rtmppx9ZHJ/file1cf0f943cf8560/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmppx9ZHJ/file1cf0f943cf8560/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
[ FAIL 0 | WARN 183 | SKIP 0 | PASS 59 ]

[ FAIL 0 | WARN 183 | SKIP 0 | PASS 59 ]
	Counter({'counted_reads': 358})
	Counter({'counted_reads': 91})
	Counter({'counted_reads': 95})
	Counter({'counted_reads': 176})
> 
> proc.time()
   user  system elapsed 
702.162  42.988 726.429